Combinatorial libraries of bicyclic guanidine derivatives and compounds therein

ABSTRACT

The invention provides a rapid approach for combinatorial synthesis and screening of combinatorial libraries of bicyclic guanidine compounds. The present invention further provides the compounds made by the combinatorial synthesis and individually as well as methods of using the same.

This application claims the benefit of U.S. Provisional Application No.60/104,594, which was converted from U.S. Ser. No. 08/794,070, filedFeb. 4, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the combinatorial synthesisof bicyclic guanidine derivatives. More specifically, the inventionprovides novel bicyclic guanidines as well as novel combinatoriallibraries comprised of many such compounds, and methods of synthesizingthe libraries.

2. Background Information

The process of discovering new therapeutically active compounds for agiven indication involves the screening of all compounds from availablecompound collections. From the compounds tested one or more structure(s)is selected as a promising lead. A large number of related analogs arethen synthesized in order to develop a structure-activity relationshipand select one or more optimal compounds. With traditional one-at-a-timesynthesis and biological testing of analogs, this optimization processis long and labor intensive. Adding significant numbers of newstructures to the compound collections used in the initial screeningstep of the discovery and optimization process cannot be accomplishedwith traditional one-at-a-time synthesis methods, except over a timeframe of months or even years. Faster methods are needed that allow forthe preparation of up to thousands of related compounds in a matter ofdays or a few weeks. This need is particularly evident when it comes tosynthesizing more complex compounds, such as the bicyclic guanidinecompounds of the present invention.

Solid-phase techniques for the synthesis of peptides have beenextensively developed and combinatorial libraries of peptides have beengenerated with great success. During the past four years there has beensubstantial development of chemically synthesized combinatoriallibraries (SCLs) made up of peptides. The preparation and use ofsynthetic peptide combinatorial libraries has been described, forexample, by Dooley in U.S. Pat. No. 5,367,053, Huebner in U.S. Pat. No.5,182,366, Appel et al. in WO PCT 92/09300, Geysen in published EuropeanPatent Application 0 138 855 and Pirrung in U.S. Pat. No. 5,143,854.Such SCLs provide the efficient synthesis of an extraordinary number ofvarious peptides in such combinatorial libraries and the rapid screeningof the library which identifies lead pharmaceutical peptides.

Peptides have been, and remain, attractive targets for drug discovery.Their high affinities and specificities toward biological receptors aswell as the ease with which large peptide libraries can becombinatorially synthesized make them attractive drug targets. Thescreening of peptide combinatorial libraries has led to theidentification of many biologically-active lead compounds. However, thetherapeutic application of peptides is limited by their poor stabilityand bioavailability in vivo. Therefore, there is a need to synthesizeand screen compounds which can maintain high affinity and specificitytoward biological receptors but which have improved pharmacologicalproperties relative to peptides.

Combinatorial approaches have recently been extended to “organic,” ornon-peptide, libraries. The organic libraries, however, are of limiteddiversity and generally relate to peptidomimetic compounds; in otherwords, organic molecules that retain peptide chain pharmacophore groupssimilar to those present in the corresponding peptide. Although thepresent invention is principally derived from the synthesis ofdipeptides, the dipeptides are substantially modified. In short, theyare chemically modified through acylation, reduction, and cyclizationinto the subject bicyclic guanidines, thus providing mixtures andindividual compounds of substantial diversity.

Significantly, many biologically active compounds contain guanidinefunctionalities. Guanidine-containing compounds have been reported to beuseful as having hypotensive and adrenergic blocking effects asdescribed, for example, in E. J. Corey and Mitsuaki Ohtani, TetrahedronLetters., 30(39):5227-5230 (1989). Guanidine-containing compounds alsocan be used as sweeteners as described, for instance, in Nagarajan etal. Synthetic Communications., 22(8):1191-1198 (1992). Because guanidinemoieties are found in many biologically active compounds and are knownto have useful therapeutic implications, there is a need to furtherstudy and develop large numbers of bicyclic guanidines and their bindingto biological receptors.

This invention satisfies these needs and provides related advantages aswell. The present invention overcomes the known limitations to classicalorganic synthesis of guanidine-containing compounds as well as theshortcomings of combinatorial chemistry with small organics orpeptidomimetics. Moreover, the present invention provides a large arrayof diverse bicyclic guanidines which can be screened for biologicalactivity, and as described below, are biologically active.

SUMMARY OF THE INVENTION

The invention provides a rapid approach for combinatorial synthesis andscreening of combinatorial libraries of bicyclic guanidine compounds.The present invention further provides individual compounds containedwithin the combinatorial library and methods of using the same, such asfor effecting analgesia. More specifically, the present inventionrelates to the generation of synthetic combinatorial libraries and oforganic compounds based on the formula:

or the formula:

wherein R¹, R², R³ and R⁴ have the meanings provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the Reaction Scheme I for preparing combinatorial librariesand compounds of the present invention.

FIG. 2 shows the Reaction Scheme II for preparing combinatoriallibraries and compounds of the present invention.

FIGS. 3A, 3B and 3C graphically depict the σ receptor assay binding datafor a bicyclic guanidine combinatorial library of the subject invention.

FIGS. 4A, 4B and 4C provide graphs depicting the κ-opioid receptorscreening data for a bicyclic guanidine combinatorial library of thesubject invention.

FIGS. 5A, 5B and 5C provide graphs depicting the antifungal activityscreening data for a bicyclic guanidine combinatorial library of thesubject invention.

FIGS. 6A, 6B and 6C provide graphs depicting the inhibition ofcalmodulin activity screening data for a bicyclic guanidinecombinatorial library of the subject invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the generation of syntheticcombinatorial libraries and individual compounds which are based on theFormula I:

In the above Formula I:

R¹ is a hydrogen atom, C₁ to C₁₀ alkyl, C₁ to C₁₀ substituted alkyl, C₇to C₁₆ phenylalkyl, C₇ to C₁₆ substituted phenylalkyl, phenyl,substituted phenyl, C₃ to C₇ cycloalkenyl, C₃ to C₇ substitutedcycloalkenyl, benzyl, or substituted benzyl;

R² is a hydrogen atom, C_(1 to C) ₁₀ alkyl, C₁ to C₁₀ substituted alkyl,C₇ to C₁₆ phenylalkyl, C₇ to C₁₆ substituted phenylalkyl, phenyl,substituted phenyl, C₃ to C₇ cycloalkyl, C₃ to C₇ substitutedcycloalkyl, benzyl, substituted benzyl, naphthyl, or substitutednaphthyl; and

R³ is a hydrogen atom, C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₁ to C₁₀substituted alkyl, C₂ to C₁₀ alkynyl, C₃ to C₇ substituted cycloalkyl,C₃ to C₇ cycloalkenyl, C₃ to C₇ substituted cycloalkenyl, C₇ to C₁₆phenylalkyl, C₇ to C₁₆ substituted phenylalkyl, C₇ to C₁₆ phenylalkenylor C₇ to C₁₆ substituted phenylalkenyl.

If desired, any one, any two or all three of the above R groups cancontain any of the above-described substituents except for a hydrogenatom.

In one embodiment of the above bicyclic guanidine combinatoriallibraries and compounds, the substituents in Formula I are as follows:

R¹ is methyl, benzyl, 2-butyl,N-methyl,N-thiocarbonylimidazole-aminobutyl, 2-methylpropyl,methylsulfinylethyl, guanidinopropyl, 2-propyl, 4-hydroxybenzyl, ethyl,dimethyl, propyl, butyl, N-methyl,N-thiocarbonylimidazole-aminopropyl,2-naphthylmethyl, cyclohexylmethyl, methylsulfonylethyl, 4-nitrobenzyl,4-chlorobenzyl, 4-fluorobenzyl,N-ethyl,N-thiocarbonylimidazole-aminobutyl, 3-pyridylmethyl, cyclohexyl,tert-butyl, N-methyl,N-thiocarbonylimidazole-4-aminobenzyl,4-ethoxybenzyl, 4-iodobenzyl, or 4-methoxybenzyl;

R² is methyl, benzyl, hydrogen, 2-butyl,N-methyl,N-thiocarbonylimidazole-aminobutyl, 2-methylpropyl,methylsulfinylethyl, guanidinopropyl, 2-propyl, 4-hydroxybenzyl, ethyl,propyl, butyl, N-methyl,N-thiocarbonylimidazole-aminopropyl,2-naphthylmethyl, cyclohexylmethyl, methylsulfonylethyl, 4-nitrobenzyl,4-chlorobenzyl, 4-fluorobenzyl,N-ethyl,N-thiocarbonylimidazole-aminobutyl, 3-pyridylmethyl, cyclohexyl,tert-butyl, N-methyl,N-thiocarbonylimidazole-4-aminobenzyl,4-ethoxybenzyl, hydroxyethyl, 4-iodobenzyl, or 4-methoxybenzyl; and

R³ is 3-phenylbutyl, m-toluylethyl, 3-fluorophenylethyl, p-toluylethyl,4-fluorophenylethyl, 3-methoxyphenylethyl, 4-methoxyphenylethyl,4-ethoxyphenylethyl, 3-(3,4-dimethoxyphenyl)propyl, 4-biphenylethyl,3,4-dimethoxyphenylethyl, phenylethyl, 3-phenylpropyl, 4-phenylbutyl,butyl, heptyl, isobutyryl, (+/−)-2-methylbutyl, isovaleryl,3-methylvaleryl, 4-methylvaleryl, (tert-butyl)ethyl, cyclohexylmethyl,cyclohexylethyl, cyclohexylbutyl, cycloheptylmethyl, 2-hydroxypropyl,ethyl, cyclobutylmethyl, cyclopentylmethyl, 3-cyclopentylpropyl,cyclohexylpropyl, 4-methyl-1-cyclohexylmethyl,4-(tert-butyl)-1-cyclohexylmethyl, 2-norbornylethyl, 1-adamantylethyl,2-ethylbutyl, 3,3-diphenylpropyl, 2-methyl-4-nitro-1-imidazolylpropyl,cyclopentylethyl, or 3-indolylethyl.

A further embodiment of the subject invention provides a combinatoriallibrary and individual compounds shown to have significant biologicalactivity, which compounds, individually or contained within thecombinatorial library, have the following substituents in Formula I:

R¹ is methyl or cyclohexyl;

R² is 4-methoxybenzyl, 2-methylpropyl or cyclohexyl; and

R³ is 3-cyclohexylpropyl or 1-adamantylethyl.

More specifically, the individual compounds are wherein (1) R¹ ismethyl, R² is 4-methoxybenzyl, and R³ is 3-cyclohexylpropyl; (2) R¹ ismethyl, R² is 4-methoxybenzyl, and R³ is 1-adamantylethyl; (3) R¹ iscyclohexyl, R² is 4-methoxybenzyl, and R³ is 1-adamantylethyl; (4) R¹ iscyclohexyl, R² is 4-methoxybenzyl, and R³ is 3-cyclohexylpropyl; (5) R¹is cyclohexyl, R² is 2-methylpropyl, and R³ is 1-adamantylethyl; (6) R¹is cyclohexyl, R² is cyclohexyl, and R³ is 1-adamantylethyl; (7) R¹ iscyclohexyl, R² is 2-methylpropyl, and R³ is 3-cyclohexylpropyl; (8) R¹is methyl, R² is cyclohexyl, and R³ is 1-adamantylethyl; (9) R¹ ismethyl, R² is cyclohexyl, and R³ is 3-cyclohexylpropyl; (10) R¹ ismethyl, R² is methylpropyl, and R³ is 1-adamantylethyl; (11) R¹ iscyclohexyl, R² is cyclohexyl and R³ is 3-cyclohexylpropyl; and (12) R¹is methyl, R² is methylpropyl, and R³ is 3-cyclohexylpropyl. The aminoacids from which these individual compounds were derived, as well as theindividual compounds described below, can be in the L- orD-configuration, resulting in the same R group, varying only in itsstereochemistry. Therefore, in the above compounds and the ones below,the R groups can be in either the R or S configuration, or a mixture ofthe two.

Additional individual compounds of the subject invention shown to havesignificant biological activity include those having the followingsubstituents in Formula I:

R¹ is benzyl or butyl;

R² is 2-naphthylmethyl, 4-ethoxybenzyl, cyclohexylmethyl,4-chlorobenzyl, 4-iodobenzyl, 4-methoxybenzyl, 4-nitrobenzyl, benzyl,cyclohexyl, N-ethyl,N-thiocarbonylimidazole-aminobutyl, or4-fluorobenzyl; and

R³ is methyl, (tert-butyl)ethyl or isovaleryl.

More preferably, seventeen individual compounds shown to havesignificant biological activity are wherein (1) R¹ is benzyl, R² is2-naphthylmethyl, R³ is methyl; (2) R¹ is benzyl, R² is 4-ethoxybenzyl,R³ is methyl; (3) R¹ is benzyl, R² is 2-naphthylmethyl, R³ is methyl;(4) R¹ is benzyl, R² is cyclohexylmethyl, R³ is methyl; (5) R¹ isbenzyl, R² is 4-chlorobenzyl, R³ is methyl; (6) R¹ is benzyl, R² is4-ethoxybenzyl, R³ is methyl; (7) R¹ is benzyl, R² is 4-iodobenzyl, R³is methyl; (8) R¹ is benzyl, R² is 4-methoxybenzyl, R³ is methyl; (9) R¹is benzyl, R² is 4-nitrobenzyl, R³ is methyl; (10) R¹ is benzyl, R² isbenzyl, R³ is (tert-butyl)ethyl; (11) R¹ is benzyl, R² is cyclohexyl, R³is methyl; (12) R¹ is benzyl, R² is 4-chlorobenzyl, R³ is methyl; (13)R¹ is benzyl, R² is benzyl, R³ is isovaleryl; (14) R¹ is benzyl, R² isN-ethyl,N-thiocarbonylimidazole-aminobutyl, R³ is methyl; (15) R¹ isbutyl, R² is benzyl, and R³ is methyl; (16) R¹ is benzyl, R² is4-fluorobenzyl, R³ is methyl; (17) R¹ is benzyl, R² is 4-fluorobenzyl,R³ is methyl.

Another embodiment of the subject invention provides a combinatoriallibrary and individual compounds shown to have significant biologicalactivity, which compounds, individually or contained within acombinatorial library, have the following substituents in Formula I:

R¹ is cyclohexyl or cyclohexylmethyl;

R² is cyclohexyl or cyclohexylmethyl; and

R³ is 4-(tert-butyl)-1-cyclohexylmethyl or 1-adamantylethyl.

More specifically, the individual compounds are wherein (1) R¹ iscyclohexyl, R² is cyclohexyl and R³ is4-(tert-butyl)-1-cyclohexylmethyl; (2) R¹ is cyclohexyl, R² iscyclohexylmethyl and R³ is 4-(tert-butyl)-1-cyclohexylmethyl; (3) R¹ iscyclohexyl, R² is cyclohexylmethyl and R³ is 1-adamantylethyl; (4) R¹ iscyclohexyl, R² is cyclohexyl and R³ is 1-adamantylethyl; (5) R¹ iscyclohexylmethyl, R² is cyclohexyl and R³ is4-(tert-butyl)-1-cyclohexylmethyl; (6) R¹ is cyclohexylmethyl, R² iscyclohexylmethyl and R³ is 4-(tert-butyl)-1-cyclohexylmethyl; (7) R¹ iscyclohexylmethyl, R² is cyclohexylmethyl and R³ is 1-adamantylethyl; and(8) R¹ is cyclohexylmethyl, R² is cyclohexyl and R³ is 1-adamantylethyl.

A further embodiment of the subject invention provides a combinatoriallibrary and individual compounds shown to have significant biologicalactivity, which compounds, individually or contained within acombinatorial library, have the following substituents in Formula I:

R¹ is cyclohexyl, cyclohexylmethyl, methyl, benzyl ormethylsulfinylethyl;

R² is cyclohexyl, cyclohexylmethyl, benzyl, hydroxyethyl,4-methoxybenzyl or 2-methylpropyl; and

R³ is 4-(tert-butyl)-1-cyclohexylmethyl, 1-adamantylethyl,cyclohexylbutyl, ethyl and 4-biphenylethyl.

More preferably, twenty-one individual compounds shown to havesignificant biological activity are wherein (1) R¹ is cyclohexylmethyl,R² is cyclohexylmethyl and R³ is 4-(tert-butyl)-1-cyclohexylmethyl; (2)R¹ is cyclohexylmethyl, R² is cyclohexylmethyl and R³ is1-adamantylethyl; (3) R¹ is cyclohexyl, R² is cyclohexylmethyl and R³ is4-(tert-butyl)-1-cyclohexylmethyl; (4) R¹ is cyclohexyl, R² iscyclohexylmethyl and R³ is 1-adamantylethyl; (5) R¹ is cyclohexylmethyl,R² is cyclohexyl and R³ is 1-adamantylethyl; (6) R¹ is cyclohexylmethyl,R² is cyclohexyl and R³ is 4-(tert-butyl)-1-cyclohexylmethyl; (7) R¹ iscyclohexyl, R² is cyclohexyl and R³ is4-(tert-butyl)-1-cyclohexylmethyl; (8) R¹ is cyclohexyl, R² iscyclohexyl and R³ is 1-adamantylethyl; and (9) R¹ is benzyl, R² ishydroxyethyl and R³ is ethyl; (10) R¹ is cyclohexyl, R² is4-methoxybenzyl and R³ is cyclohexylbutyl; (11) R¹ is cyclohexyl, R² is4-methoxybenzyl and R³ is 1-adamantylethyl; (12) R¹ is cyclohexyl, R² is4-methoxybenzyl and R³ is cyclohexylbutyl; (13) R¹ is cyclohexyl, R² iscyclohexylmethyl and R³ is cyclohexylbutyl; (14) R¹ is benzyl, R² isbenzyl and R³ is 4-biphenylethyl; (15) R¹ is cyclohexyl, R² is2-methylpropyl and R³ is 1-adamantylethyl; (16) R¹ is benzyl, R² isbenzyl and R³ is 1-adamantylethyl; (17) R¹ is benzyl, R² is benzyl andR³ is cyclohexylbutyl; (18) R¹ is cyclohexyl, R² is 2-methylpropyl andR³ is cyclohexylbutyl; (19) R¹ is benzyl, R² is benzyl and R³ is4-(tert-butyl)-1-cyclohexylmethyl; (20) R¹ is methyl, R² is benzyl andR³ is ethyl; and (21) R¹ is methylsulfinylethyl, R² is benzyl and R³ isethyl.

Additional individual compounds shown to have significant biologicalactivity include those having the following substituents in Formula I:

R¹ is benzyl or N-(methyl)indol-3-ylmethyl;

R² is benzyl or indol-3-ylmethyl; and

R³ is 2,4 dinitrobenzyl or ethyl.

More preferably, three individual compounds shown to have significantbiological activity are wherein (1) R¹ is benzyl, R² is benzyl and R³ is2,4 dinitrobenzyl; (2) R¹ is benzyl, R² is indol-3-ylmethyl and R³ isethyl; (3) R¹ is N-(methyl)indol-3-ylmethyl, R² is benzyl and R³ isethyl.

The present invention also relates to the generation of syntheticcombinatorial libraries and individual compounds which are based on theFormula II:

In the above Formula II:

R¹ is a hydrogen atom, C₁ to C₁₀ alkyl, C₁ to C₁₀ substituted alkyl, C₇to C₁₆ phenylalkyl, C₇ to C₇ to C₁₆ substituted phenylalkyl, phenyl,substituted phenyl, C₃ to C₇ cycloalkyl, C₃ to C₇ substitutedcycloalkyl, benzyl, or substituted benzyl;

R² is a hydrogen atom, C₁ to C₁₀ alkyl, C₁ to C₁₀ substituted alkyl, C₇to C₁₆ phenylalkyl, C₇ to C₁₆ substituted phenylalkyl, phenyl,substituted phenyl, C₃ to C₇ cycloalkyl, C₃ to C₇ substitutedcycloalkyl, benzyl, or substituted benzyl;

R³ is a hydrogen atom, C₁ to C₁₀ alkyl, C₁ to C₁₀ substituted alkyl, C₇to C₁₆ phenylalkyl, C₇ to C₁₆ substituted phenylalkyl, phenyl,substituted phenyl, C₃ to C₇ cycloalkyl, C₃ to C₇ substitutedcycloalkyl, benzyl, or substituted benzyl; and

R⁴ is a hydrogen atom, C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₁ to C₁₀substituted alkyl, C₂ to C₁₀ alkynyl, C₃ to C₇ substituted cycloalkyl,C₃ to C₇ cycloalkenyl, C₃ to C₇ substituted cycloalkenyl, C₇ to C₁₆phenylalkyl, C₇ to C₁₆ substituted phenylalkyl, C₇ to C₁₆ phenylalkenylor C₇ to C₁₆ substituted phenylalkenyl.

If desired, any one, any two, any three or all four of the above Rgroups can contain any of the above-described substituents except for ahydrogen atom.

In one embodiment of the above bicyclic guanidine combinatoriallibraries and compounds, the substituents in Formula II are as follows:

R¹ is methyl, benzyl, 2-butyl, 2-methylpropyl, 2-propyl,2-bromobenzyloxycarbonylbenzyl, ethyl, dimethyl, propyl, butyl,2-napthylmethyl, cyclohexylmethyl, 4-fluorobenzyl, 4-chlorobenzyl,cyclohexyl, 4-ethoxybenzyl, 4-iodobenzyl, or 4-methoxybenzyl;

R² is methyl, benzyl, 2-butyl, 2-methylpropyl, 2-propyl,2-bromobenzyloxycarbonylbenzyl, ethyl, propyl, butyl, 2-naphthylmethyl,methylsulfonylethyl, cyclohexylmethyl, 4-fluorobenzyl, 4-chlorobenzyl,cyclohexyl, 4-ethoxybenzyl, 4-iodobenzyl, or 4-methoxybenzyl;

R³ is methyl, benzyl, hydrogen, 2-methylpropyl, propyl, butyl,cyclohexylmethyl, 4-ethoxybenzyl, or 4-methoxybenzyl; and

R⁴ is 1-phenyl-1-cyclopropyl, 1-phenylpropyl, 2-phenylpropyl, m-xylyl,3-fluorobenzyl, 3-bromobenzyl, 3-trifluoromethylbenzyl, p-xylyl,3-methoxybenzyl, 4-bromobenzyl, 4-methoxybenzyl, 4-ethoxybenzyl,1-(4-isobutylphenyl)ethyl, 3,4-dichlorobenzyl, 3-(3,4-dimethoxy)ethyl,4-biphenylmethyl, 1-phenylpropen-2-yl, 2-trifluoromethylstryl,3,4-dimethoxybenzyl, 3,4-dihydroxybenzyl, 2-methoxystyryl, phenyl,4-chlorostyryl, 3-methoxyphenyl, 4-isopropylphenyl, 4-vinylphenyl,4-fluorophenyl, 4-bromophenyl, 3,4-dimethoxystyryl, trans-styryl,3,4-dimethylphenyl, 3-fluoro-4-methylphenyl, 3-bromo-4-methylphenyl,3-iodo-4-methylphenyl, 3,4-dichlorophenyl, 4-biphenyl,3,4-difluorophenyl, m-tolyl, benzyl, phenethyl,3-methoxy-4-methylphenyl, 3-phenylpropyl, 3,4-dimethoxyphenyl,4-ethyl-4′-biphenyl, 3,4,5-trimethoxyphenyl, propyl, hexyl, 2-propyl,(+/−)-2-butyl, isobutyl, 2-methylbutyl, isovaleryl, p-tolyl, p-anisyl,cyclohexyl, cyclohexylmethyl, cyclohexylpropyl, cycloheptyl, methyl,2-methylcyclopropyl, cyclobutyl, cyclopentyl, cyclopentylethyl, 2-furyl,cyclohexylethyl, 4-methylcyclohexyl, 4-t-butylcyclohexyl,4-methylcyclohexylmethyl, but-2-en-1-yl, 2-norbornylmethyl, or2-thienyl.

Because combinatorial libraries can be screened while still bound toresin, additional embodiments of the invention include any of the abovedescribed combinatorial libraries bound to a solid-phase resin. Thecompounds in such libraries would be resin-bound through the iminenitrogen in the above Formulae and, therefore, the guanidine would bepositively charged while bound to the resin. The resins to which suchcompounds can be bound are functionalized amine resins, solid-phaseresins cross-linked with amino groups, in which case it would beappreciated by those in the art that the amine function can be cleavedfrom the resin during standard hydrogen fluoride (HF) cleavageprocedures and retained with the subject compounds.

In the above Formulae the stereochemistry of the relevant chiral R¹through R⁴ groups can independently be in the R or S configuration, or amixture of the two. For instance, as will be described in further detailbelow the R¹ and R² groups in Formula I and the R¹, R² and R³ groups inFormula II are the side chains of the α-carbon of various amino acids.The amino acids can be in the L- or D-configuration, resulting in thesame R group, varying only in its stereochemistry.

In the above Formulae, the term “C₁ to C₁₀ alkyl” denotes such radicalsas methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl,amyl, tert-amyl, hexyl, heptyl and the like. A preferred “C₁ to C₁₀alkyl” group is methyl.

The term “C₂ to C₁₀ alkenyl” denotes such radicals as vinyl, allyl,2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl,3-hexenyl, 4-hexenyl, 5-hexenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl,5-heptenyl, 6-heptenyl, as well as dienes and trienes of straight andbranched chains.

The term “C₂ to C₁₀ alkynyl” denotes such radicals as ethynyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, as well as di- and tri-ynes.

The term “substituted” as it is used in “C₁ to C₁₀ substituted alkyl,”“C₂ to C₁₀ substituted alkenyl,” and “C₂ to C₁₀ substituted alkynyl,”denotes that the above C₁ to C₁₀ alkyl groups and C₂ to C₁₀ alkenyl andalkynyl groups are substituted by one or more, and preferably one ortwo, halogen, hydroxy, protected hydroxy, C₃ to C₇ cycloalkyl, C₃ to C₇substituted cycloalkyl, naphthyl, substituted naphthyl, adamantyl,abietyl, thiofuranyl, indolyl, substituted indolyl, norbornyl, amino,protected amino, (monosubstituted)amino, protected(monosubstituted)amino, (disubstituted)amino, guanidino,(monosubstituted)guanidino, (disubstituted)guanidino,(trisubstituted)guanidino, imidazolyl, pyrolidinyl, C₁ to C₇ acyloxy,nitro, heterocycle, substituted heterocycle, C₁ to C₄ alkyl ester,carboxy, protected carboxy, carbamoyl, carbamoyloxy, carboxamide,protected carboxamide, cyano, methylsulfonylamino, methylsulfinyl,methylsulfonyl, sulfurhydryl, C₁ to C₄ alkylthio, C₁ to C₄ alkylsulfonyl or C₁ to C₄ alkoxy groups. The substituted alkyl groups may besubstituted once or more, and preferably once or twice, with the same orwith different substituents.

Examples of the above substituted alkyl groups include the cyanomethyl,nitromethyl, chloromethyl, hydroxymethyl, tetrahydropyranyloxymethyl,trityloxymethyl, propionyloxymethyl, aminomethyl, carboxymethyl,allyloxycarbonylmethyl, allylcaroxybonylaminomethyl, carbamoyloxymethyl,methoxymethyl, ethoxymethyl, t-butoxymethyl, acetoxymethyl,chloromethyl, bromomethyl, iodomethyl, 6-hydroxyhexyl,2,4-dichloro(n-butyl), 2-amino(iso-propyl), 2-carbamoyloxyethyl,chloroethyl, bromoethyl, fluoroethyl, iodoethyl, chloropropyl,bromopropyl, fluoropropyl, iodopropyl and the like.

In preferred embodiments of the subject invention, preferred groupsinclude C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₂ to C₁₀ alkynyl, C₁ toCsubstituted alkyl, C₂ to C₁₀ substituted alkenyl, or C₂ to C₁₀substituted alkynyl and, regarding alkyl or substituted alkyl groups,more preferably C₁ to C₇, and even more preferably, C₁ to C₆. However,it would be appreciated to those of skill in the art that one or a fewcarbons could be added to an alkyl, alkenyl, alkynyl, substituted orunsubstituted, without substantially modifying the structure andfunction of the subject compounds and that, therefore, such additionswould not depart from the spirit of the invention.

The term “C₁ to C₄ alkoxy” as used herein denotes groups such asmethoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy and likegroups. A preferred C₁ to C₄ alkoxy group is methoxy.

The term “C₁ to C₁ acyloxy” denotes herein groups such as formyloxy,acetoxy, propanoyloxy, butanoyloxy, pentanoyloxy, hexanoyloxy,heptanoyloxy, and the like.

Similarly, the term “C₁ to C₇ acyl” encompasses groups such as formyl,acetyl, propionoyl, butyroyl, pentanoyl, hexanoyl, heptanoyl, benzoyland the like.

The substituent term “C₃ to C₇ cycloalkyl” includes the cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings. Thesubstituent term “C₃ to C₇ substituted cycloalkyl” indicates the abovecycloalkyl rings substituted by a halogen, hydroxy, protected hydroxy,phenyl, substituted phenyl, heterocycle, substituted heterocycle, C₁ toC₁₀ alkyl, C₁ to C₄ alkoxy, carboxy, protected carboxy, amino, orprotected amino.

The substituent term “C₃ to C₇ cycloalkenyl” indicates a 1,2, or3-cyclopentenyl ring, a 1,2,3 or 4-cyclohexenyl ring or a 1,2,3,4 or5-cycloheptenyl ring, while the term “C₃ to C₇ substituted cycloalkenyl”denotes the above C₃ to C₇ cycloalkenyl rings substituted by a C₁ to C₁₀alkyl radical, halogen, hydroxy, protected hydroxy, C₁ to C₄ alkoxy,carboxy, protected carboxy, amino, or protected amino.

The term “heterocyclic ring” or “heterocycle” denotes optionallysubstituted five-membered or six-membered rings that have 1 to 4heteroatoms, such as oxygen, sulfur and/or nitrogen, in particularnitrogen, either alone or in conjunction with sulfur or oxygen ringatoms. These five-membered or six-membered rings may be fullyunsaturated or partially unsaturated, with fully unsaturated rings beingpreferred. Preferred heterocyclic rings include pyridinyl, pyrimidinyl,pyrazinyl, furanyl, imidazolyl and thiofuranyl rings. The heterocylescan be substituted or unsubstituted as, for example, with suchsubstituents as those described in relation to substituted phenyl orsubstituted naphthyl.

The term “C₇ to C₁₆ phenylalkyl” denotes a C₁ to C₁₀ alkyl groupsubstituted at any position by a phenyl ring. Examples of such a groupinclude benzyl, 2-phenylethyl, 3-phenyl-(n-prop-1-yl),4-phenyl-(-hex-1-yl), 3-phenyl-(n-am-2-yl), 3-phenyl-(sec-butyl), andthe like. A preferred group is the benzyl group.

The term “C₇ to C₁₆ substituted phenylalkyl” denotes a C₇ to C₁₆arylalkyl group substituted on the C₁ to C₁₀ alkyl portion with one ormore, and preferably one or two, groups chosen from halogen, hydroxy,protected hydroxy, keto, C₂ to C₃ cyclic ketal, phenyl, amino, protectedamino, C₁ to C₇ acyloxy, nitro, carboxy, protected carboxy, carbamoyl,carbamoyloxy, cyano, N-(methylsulfonylamino) or C₁ to C₄ alkoxy; and/orthe phenyl group may be substituted with 1 or 2 groups chosen fromhalogen, hydroxy, protected hydroxy, nitro, C₁ to C₁₀ alkyl, C₁ to C₆substituted alkyl, C₁ to C₄ alkoxy, carboxy, protected carboxy,carboxymethyl, protected carboxymethyl, hydroxymethyl, protectedhydroxymethyl, aminomethyl, protected aminomethyl, amino,(monosubstituted)amino, (disubstituted)amino, a N-(methylsulfonylamino)group, or a phenyl group, substituted or unsubstituted, for a resultingbiphenyl group. When either the C₁ to C₁₀ alkyl portion or the phenylportion or both are mono- or di-substituted the substituents can be thesame or different.

Examples of the term “C₇ to C₁₆ substituted phenylalkyl” include groupssuch as 2-phenyl-1-chloroethyl, 2-(4-methoxyphenyl)eth-1-yl,2,6-dihydroxy-4-phenyl(n-hex-2-yl),5-cyano-3-methoxy-2-phenyl(n-pent-3-yl),3-(2,6-dimethylphenyl)n-prop-1-yl, 4-chloro-3-aminobenzyl,6-(4-methoxyphenyl)-3-carboxy(n-hex-1-yl),5-(4-aminomethyl-phenyl)-3-(aminomethyl)(n-pent-2-yl),5-phenyl-3-keto-(n-pent-1-yl),4-(4-aminophenyl)-4-(1,4-oxetanyl)(n-but-1-yl), and the like.

The term “C₇ to C₁₆ phenylalkenyl” denotes a C₁ to C₁₀ alkenyl groupsubstituted at any position by a phenyl ring. The term “C₇ to C₁₆substituted phenylalkenyl” denotes a C₇ to C₁₆ arylalkyl groupsubstituted on the C₁ to C₁₀ alkenyl portion. Substituents can the sameas those as defined above in relation to C₇ to C₁₆ phenylalkyl and C₇ toC₁₆ substituted phenylalkyl.

The term “substituted phenyl” specifies a phenyl group substituted withone or more, and preferably one or two, moieties chosen from the groupsconsisting of halogen, hydroxy, protected hydroxy, cyano, nitro, C₁ toC₁₀ alkyl, C₁ to C₁₀ substituted alkyl, C₁ to C₄ alkoxy, carboxy,protected carboxy, carboxymethyl, protected carboxymethyl,hydroxymethyl, protected hydroxymethyl, amino, protected amino,(monosubstituted)amino, protected (monosubstituted)amino,(disubstituted)amino, trifluoromethyl, N-(methylsulfonylamino), orphenyl, substituted or unsubstituted, such that, for example, a biphenylresults.

Examples of the term “substituted phenyl” include a mono- ordi(halo)phenyl group such as 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3-chlorophenyl, 3-bromophenyl,4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl,2-fluorophenyl and the like; a mono or di(hydroxy)phenyl groups such as4-hydroxyphenyl, 3-hydroxyphenyl, 2,4-dihydroxyphenyl, theprotected-hydroxy derivatives thereof and the like; a nitrophenyl groupsuch as 3-or 4-nitrophenyl; a cyanophenyl group for example,4-cyanophenyl; a mono- or di(lower alkyl)phenyl group such as4-methylphenyl, 2,4-dimethylphenyl, 2-methylphenyl,4-(iso-propyl)phenyl, 4-ethylphenyl, 3-(n-prop-1-yl)phenyl and the like;a mono or di(alkoxyl)phenyl group, for example, 2,6-dimethoxyphenyl,4-methoxyphenyl, 3-ethoxyphenyl, 4-(isopropoxy)phenyl,4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl, 3-(4-methylphenoxy)phenyl,and the like,; 3-or 4-trifluoromethylphenyl; a mono- or dicarboxyphenylor (protected carboxy)phenyl group such as 4-carboxyphenyl or2,4-di(protected carboxy)phenyl; a mono-or di(hydroxymethyl)phenyl or(protected hydroxymethyl)phenyl such as 3-(protectedhydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; a mono- ordi(aminomethyl)phenyl or (protected aminomethyl)phenyl such as2-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or a mono-or di(N-(methylsulfonylamino))phenyl such as3-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups wherein the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl, 2-hydroxy4-chlorophenyl and the like.

The term “substituted benzyl” means a benzyl group substituted with oneor more, and preferably one or two, moieties chosen from the same groupsas provided with reference to “substituted phenyl.” Examples ofsubstituted benzyl include 4-bromobenzyl, 4-chlorobenzyl,4-fluorobenzyl, 4-ethoxybenzyl, 4-hydroxybenzyl, 4-iodobenzyl, and thelike.

The term “substituted naphthyl” specifies a naphthyl group substitutedwith one or more, and preferably one or two, moieties chosen from thegroups consisting of halogen, hydroxy, protected hydroxy, cyano, nitro,C₁ to C₁₀ alkyl, C₁ to C₄ alkoxy, carboxy, protected carboxy,carboxymethyl, protected carboxymethyl, hydroxymethyl, protectedhydroxymethyl, amino, protected amino,(monosubstituted)amino, protected(monosubstituted)amino, (disubstituted)amino trifluoromethyl orN-(methylsulfonylamino). Examples of substituted naphthyl include2-(methoxy)-naphthyl and 4-(methoxy)naphthyl.

The term “substituted indolyl” specifies a indolyl group substituted,either at the nitrogen or carbon, or both, with one or more, andpreferably one or two, moieties chosen from the groups consisting ofhalogen, hydroxy, protected hydroxy, cyano, nitro, C₁ to C₁₀ alkyl, C₁to C₁₀ substituted alkyl, C₁ to C₁₀ alkenyl, C₇ to C₁₆ phenylalkyl, C₇to C₁₆ substituted phenylalkyl, C₁ to C₆ alkoxy, C₁ to C₇ acyl, alkylcarboxy, protected carboxy, carboxymethyl, protected carboxymethyl,hydroxymethyl, protected hydroxymethyl, formyl, amino, protected amino,monosubstituted amino, or disubstituted amino.

Examples of the term “substituted indolyl” include such groups as6-fluoro, 5-fluoro, 5-bromo, 5-hydroxy, 5-methyl, 6-methyl, 7-methyl,1-methyl, 1-ethyl, 1-benzyl, 1-napth-2-ylmethyl, and the like. Anexample of a disubstituted indolyl is 1-methyl-5-methyl indolyl.

The terms “halo” and “halogen” refer to the fluoro, chloro, bromo oriodo groups.

The term “(monosubstituted)amino” refers to an amino group with onesubstituent chosen from the groups consisting of phenyl, substitutedphenyl, C₁ to C₁₀ alkyl, and C₇ to C₁₆ arylalkyl, wherein the latterthree substituent terms are as defined above. The (monosubstituted)aminocan additionally have an amino-protecting group as encompassed by theterm “protected (monosubstituted)amino.”

The term “(disubstituted)amino” refers to amino groups with twosubstituents chosen from the group consisting of phenyl,thiocarbonylimidazole, substituted phenyl, C₁ to C₁₀ alkyl, and C₇ toC₁₆ arylalkyl wherein the latter three substituent terms are asdescribed above. The two substituents can be the same or different.

The terms “(monosubstituted)guanidino,” “(disubstituted)guanidino,” and“(trisubstituted)guanidino” are where the guanidino groups issubstituted with one, two, or three substituents, respectively. Thesubstituents can be any of those as defined above in relation to(monosubstituted)amino and (disubstituted)amino and, where more than onesubstituent is present, the substituents can be the same or different.

The term “amino-protecting group” as used herein refers to substituentsof the amino group commonly employed to block or protect the aminofunctionality while reacting other functional groups on the aminecomponent. The term “protected (monosubstituted)amino” means there is anamino-protecting group on the monosubstituted amino nitrogen atom. Inaddition, the term “protected carboxamide” means there is anamino-protecting group replacing the proton so that there is noN-alkylation. Examples of such amino-protecting groups include theformyl (“For”) group, the trityl group (Trt), the phthalimido group, thetrichloroacetyl group, the chloroacetyl, bromoacetyl, and iodoacetylgroups, urethane-type blocking groups, such as t-butoxy-carbonyl(“Boc”), 2-(4-biphenylyl)propyl(2)oxycarbonyl (“Bpoc”),2-phenylpropyl(2)oxycarbonyl (“Poc”), 2-(4-xenyl)isopropoxycarbonyl,1,1-diphenylethyl(1)-oxycarbonyl, 1,1-diphenylpropyl(1)oxycarbonyl,2-(3,5-dimethoxyphenyl)propyl(2)oxycarbonyl (“Ddz”),2-(p-toluyl)propyl(2)oxycarbonyl, cyclopentanyloxycarbonyl,1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl,1-methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl,2-(4-toluylsulfonyl)ethoxycarbonyl, 2-(methylsulfonyl)ethoxycarbonyl,2-(triphenylphosphine)ethoxycarbonyl, 9-fluoroenylmethoxycarbonyl(“Fmoc”), 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl,1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl,5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-ethynyl(2)propoxycarbonyl,cyclopropylmethoxycarbonyl, isobornyloxycarbonyl,1-piperidyloxycarbonyl, benzyloxycarbonyl (“Z”),4-phenylbenzyloxycarbonyl, 2-methylbenzyloxycarbonyl,α-2,4,5,-tetramethylbenzyloxycarbonyl (“Tmz”),4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl,4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl,2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl,4-(decyloxy)benzyloxycarbonyl, and the like; the benzoylmethylsulfonylgroup, dithiasuccinoyl (“Dts”), the 2-(nitro)phenylsulfenyl group(“Nps”), the diphenylphosphine oxide group, and like amino-protectinggroups. The species of amino-protecting group employed is not criticalso long as the derivatized amino group is stable to the conditions ofthe subsequent reaction(s) and can be removed at the appropriate pointwithout disrupting the remainder of the compounds. Preferredamino-protecting groups are Boc and Fmoc. Further examples ofamino-protecting groups embraced to by the above term are well known inorganic synthesis and the peptide art and are described by, for example,T. W. Greene and P. G. M. Wuts, “Protective Groups in OrganicSynthesis,” 2nd ed., John Wiley and Sons, New York, N.Y., 1991, Chapter7, M. Bodanzsky, “Principles of Peptide Synthesis,” 1st and 2nd reviseded., Springer-Verlag, New York, N.Y., 1984 and 1993, and Stewart andYoung, “Solid Phase Peptide Synthesis,” 2nd ed., Pierce Chemical Co.,Rockford, Ill., 1984, each of which is incorporated herein by reference.The related term “protected amino” defines an amino group substitutedwith an amino-protecting group discussed above.

The term “carboxy-protecting group” as used herein refers to one of theester derivatives of the carboxylic acid group commonly employed toblock or protect the carboxylic acid group while reactions are carriedout on other functional groups on the compound. Examples of suchcarboxylic acid protecting groups include 4-nitrobenzyl,4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl,2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl, pentamethylbenzyl,3,4-methylenedioxybenzyl, benzhydryl, 4,4′-dimethoxytrityl,4,4′,4″-timethoxytrityl, 2-phenylprop-2-yl, trimethylsilyl,t-butyldimethylsilyl, 2,2,2-trichloroethyl, β-(trimethylsilyl)ethyl,β-(di(n-butyl)methylsilyl)ethyl, p-toluenesulfonylethyl,4-nitrobenzyl-sulfonylethyl, allyl, cinnamyl,1-(trimethylsilylmethyl)-prop-1-en-3-yl, and like moieties. The speciesof carboxy-protecting group employed is not critical so long as thederivatized carboxylic acid is stable to the conditions of subsequentreaction(s) and can be removed at the appropriate point withoutdisrupting the remainder of the molecule. Further examples of thesegroups are found in E. Haslam, “Protective Groups in Organic Chemistry,”J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, andT. W. Greene and P. G. M. Wuts, “Protective Groups in OrganicSynthesis,” 2nd ed., John Wiley and Sons, New York, N.Y., 1991, Chapter5, each of which is incorporated herein by reference. A related term is“protected carboxy,” which refers to a carboxy group substituted withone of the above carboxy-protecting groups.

The term “hydroxy-protecting group” refers to readily cleavable groupsbonded to hydroxyl groups, such as the tetrahydropyranyl,2-methoxyprop-2-yl, 1-ethoxyeth-1-yl, methoxymethyl,β-methoxyethoxymethyl, methylthiomethyl, t-butyl, t-amyl, trityl,4-methoxytrityl, 4,4′-dimethoxytrityl, 4,4′,4″-trimethoxytrityl, benzyl,allyl, trimethylsilyl, (t-butyl)dimethylsilyl and2,2,2-trichloroethoxycarbonyl groups and the like. The species ofhydroxy-protecting groups is not critical so long as the derivatizedhydroxyl group is stable to the conditions of subsequent reaction(s) andcan be removed at the appropriate point without disrupting the remainderof the bicyclic guanidine. Further examples of hydroxy-protecting groupsare described by C. B. Reese and E. Haslam, “Protective Groups inOrganic Chemistry,” J. G. W. McOmie, Ed., Plenum Press, New York, N.Y.,1973, Chapters 3 and 4, respectively, and T. W. Greene and P. G. M.Wuts, “Protective Groups in Organic Synthesis,” 2nd ed., John Wiley andSons, New York, N.Y., 1991, Chapters 2 and 3.

The substituent term “C₁ to C₄ alkylthio” refers to sulfide groups suchas methylthio, ethylthio, n-propylthio, iso-propylthio, n-butylthio,t-butylthio and like groups.

The term “C₁ to C₄ alkylsulfonyl” encompasses groups such asmethylsulfonyl, ethylsulfonyl, n-propylsulfonyl, iso-propylsulfonyl,n-butylsulfonyl, t-butylsulfonyl, and the like.

Phenylthio, phenyl sulfoxide, and phenylsulfonyl compounds are known inthe art and these terms have their art recognized definition. By“substituted phenylthio,” “substituted phenyl sulfoxide,” and“substituted phenylsulfonyl” is meant that the phenyl can be substitutedas described above in relation to “substituted phenyl.”

The substituent terms “cyclic C₂ to C₁₀ alkylene,” “substituted cyclicC₂ to C₁₀ alkylene,” “cyclic C₂ to C₁₀ heteroalkylene,” and “substitutedcyclic C₂ to C₁₀ heteroalkylene,” defines such a cyclic group bonded(“fused”) to the phenyl radical. The cyclic group may be saturated orcontain one or two double bonds. Furthermore, the cyclic group may haveone or two methylene groups replaced by one or two oxygen, nitrogen orsulfur atoms.

The cyclic alkylene or heteroalkylene group may be substituted once ortwice by substituents selected from the group consisting of thefollowing moieties: hydroxy, protected hydroxy, carboxy, protectedcarboxy, keto, ketal, C₁ to C₄ alkoxycarbonyl, formyl, C₂ to C₄alkanoyl, C₁ to C₁₀ alkyl, carbamoyl, C₁ to C₄ alkoxy, C₁ to C₄alkylthio, C₁ to C₄ alkylsulfoxide, C₁ to C₄ alkylsulfonyl, halo, amino,protected amino, hydroxymethyl or a protected hydroxymethyl.

The substituent term “C₁ to C₄ alkylsulfoxide” indicates sulfoxidegroups such as methylsulfoxide, ethylsulfoxide, n-propylsulfoxide,iso-propylsulfoxide, n-butylsulfoxide, sec-butylsulfoxide, and the like.

The cyclic alkylene or heteroalkylene group fused onto the benzeneradical can contain two to ten ring members, but it preferably containsfour to six members. Examples of such saturated cyclic groups are whenthe resultant bicyclic ring system is 2,3-dihydroindanyl and a tetralinring. When the cyclic groups are unsaturated, examples occur when theresultant bicyclic ring system is a naphthyl ring or indanyl. An exampleof a cyclic group which can be fused to a phenyl radical which has twooxygen atoms and which is fully saturated is dioxanyl. Examples of fusedcyclic groups which each contain one oxygen atom and one or two doublebonds are when the phenyl ring is fused to a furo, pyrano,dihydrofurano, or dihydropyrano ring. Examples of cyclic groups whicheach have one nitrogen atom and contain one or two double more doublebonds are when the phenyl is fused to a pyridino or pyrano ring. Anexample of a fused ring system having one nitrogen and two phenylradicals is a carbozoyl group. Examples of cyclic groups which each haveone sulfur atom and contain one or two double bonds are when the phenylis fused to a thieno, thiopyrano, dihydrothieno or dihydrothiopyranoring. Examples of cyclic groups which contain two heteroatoms selectedfrom sulfur and nitrogen and one or two double bonds are when the phenylring is fused to a thiazolo, isothiazolo, dihydrothiazolo ordihydroisothiazolo ring. Examples of cyclic groups which contain twoheteroatoms selected from oxygen and nitrogen and one or two doublebonds are when the benzene ring is fused to an oxazolo, isoxazolo,dihydrooxazolo or dihydroisoxazolo ring. Examples of cyclic groups whichcontain two nitrogen heteroatoms and one or two double bonds occur whenthe benzene ring is fused to a pyrazolo, imidazolo, dihydropyrazolo ordihydroimidazolo ring.

One or more of the bicyclic guanidines within a given combinatoriallibrary may be present as a pharmaceutically acceptable salt. The term“pharmaceutically-acceptable salt” encompasses those salts that formwith the carboxylate anions and include salts formed with the organicand inorganic cations discussed below. Furthermore, the term includessalts that form by standard acid-base reactions with basic groups (suchas amino groups) and organic or inorganic acids. Such acids includehydrochloric, sulfuric, phosphoric, acetic, succinic, citric lactic,maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic,d-camphoric, glutaric, phthalic, tartaric, lauric, stearic, salicyclic,methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, andlike acids.

The term “organic or inorganic cation” refers to counterions for thecarboxylate anion of a carboxylate salt. The counter-ions are chosenfrom the alkali and alkaline earth metals, (such as lithium, sodium,potassium, barium and calcium); ammonium; and the organic cations (suchas dibenzylammonium, benzylammonium, 2-hydroxyethylammonium,bis(2-hydroxyethyl)ammonium, phenylethylbenzylammonium,dibebenzylethylenediammonium, and like cations). Other cationsencompassed by the above term include the protonated form of procaine,quinine and N-methylglucosamine, and the protonated forms of basic aminoacids such as glycine, ornithine, histidine, phenylglycine, lysine andarginine. Furthermore, any zwitterionic form of the instant compoundsformed by a carboxylic acid and an amino group is referred to by thisterm. A preferred cation for the carboxylate anion is the sodium cation.

The compounds of the above Formulae can also exist as solvates andhydrates. Thus, these compounds may crystallize with, for example,waters of hydration, or one, a number of, or any fraction thereof ofmolecules of the mother liquor solvent. The solvates and hydrates ofsuch compounds are included within the scope of this invention.

One or more bicyclic guanidines can be in the biologically active esterform, such as the non-toxic, metabolically-labile ester-form. Such esterforms induce increased blood levels and prolong the efficacy of thecorresponding non-esterified forms of the compounds. Ester groups whichcan be used include the lower alkoxymethyl groups, for example,methoxymethyl, ethoxymethyl, iso-propoxymethyl and the like; the α-(C₁to C₄) alkoxyethyl groups, for example methoxyethyl, ethoxyethyl,propxyethyl, iso-propoxyethyl, and the like; the2-oxo-1,3-dioxolen-4-ylmethyl groups, such as5-methyl-2-oxo-1,3-dioxolen-4-ylmethyl,5-phenyl-2-oxo-1,3-dioxolen-4-ylmethyl, and the like; the C₁ to C₃alkylthiomethyl groups, for example methylthiomethyl, ethylthiomethyl,iso-propylthiomethyl, and the like; the acyloxymethyl groups, forexample pivaloyloxymethyl, pivaloyloxyethyl, α-acetoxymethyl, and thelike; the ethoxycarbonyl-1-methyl group; the α-acetoxyethyl; the3-phthalidyl or 5,6-dimethylphthalidyl groups; the 1-(C₁ to C₄alkyloxycarbonyloxy)ethyl groups such as the 1-(ethoxycarbonyloxy)ethylgroup; and the 1-(C₁ to C₄ alkylaminocarbonyloxy)ethyl groups such asthe 1-(methylaminocarbonyloxy)ethyl group.

As used herein, a “combinatorial library” is an intentionally createdcollection of differing molecules which can be prepared by the syntheticmeans provided below or otherwise and screened for biological activityin a variety of formats (e.g., libraries of soluble molecules, librariesof compounds attached to resin beads, silica chips or other solidsupports). A “combinatorial library,” as defined above, involvessuccessive rounds of chemical syntheses based on a common startingstructure. The combinatorial libraries can be screened in any variety ofassays, such as those detailed below as well as others useful forassessing the biological activity of bicyclic guanidines. Thecombinatorial libraries will generally have at least one active compoundand are generally prepared such that the compounds are in equimolarquantities.

Compounds disclosed in previous work that are not in a mixture are notpart of a “combinatorial library” of the invention (see, for example,Wellner et al., DE 30 18 023 A1 (1981)). In addition, compounds that arein an unintentional or undesired mixture are not part of a“combinatorial library” of the invention.

A combinatorial library of the invention can contain two or more of theabove-described bicyclic guanidine compounds. The invention furtherprovides a combinatorial library containing five or more of theabove-described bicyclic guanidine compounds. In another embodiment ofthe invention, a combinatorial library can contain ten or more of theabove-described bicyclic guanidine compounds. In yet another embodimentof the invention, a combinatorial library can contain fifty or more ofthe above-described bicyclic guanidine compounds. If desired, acombinatorial library of the invention can contain 100,000 or more, oreven 1,000,000 or more, of the above-described bicyclic guanidinecompounds.

As will be described in further detail, one combinatorial library wasprepared with the structure of Formula I where the R¹, R² and R³positions varied as described above and, in further detail, below.Moreover, as will be described in further detail, another combinatoriallibrary was prepared with the structure of Formula II where the R¹, R²,R³ and R⁴ positions varied as described above and, in further detail,below. It should be appreciated, however, that such combinatoriallibraries can comprise several smaller “sub-libraries” or sets ofmixtures of compounds, depending on the format of preparation and thevarying R groups. Sublibraries are described in further detail below.

The bicyclic guanidine combinatorial library and compounds of Formula Ican be prepared according to the general Reaction Scheme I in FIG. 1.The combinatorial libraries were prepared using solid-phase techniques.The solid-phase resin, here p-methylbenzhydrylamine resin (MBHA), isindicated in FIG. 1 by the large circle and dash. After the addition ofa first protected amino acid (having side chain R¹) to the resin, theresin-bound amino acid is deprotected. Following neutralization, asecond protected amino acid (having side chain R²) is added usingtraditional solid phase peptide chemistry. Following amino deprotection,the resulting dipeptide is then acylated with one of a wide range ofavailable carboxylic acids to obtain the acylated dipeptide. Exemplaryamino acids and carboxylic acids are discussed in detail below.

The next key step in the synthetic process, as shown in FIG. 1, is thereduction of the amide groups of the acylated dipeptide using diboranein THF at 65° C. to generate three secondary amines. This method hasbeen used to generate diverse chemical libraries using the “librariesfrom libraries” concept as described, for instance, in Ostresh et al.Proc. Nat. Acad. Sci., 91:11138 (1994) and Cuervo et al. In Peptides,1994, Proceedings of the 23rd European Peptide Symposium (Maia, H. L. S,ed): 465-466 (1995), each of which are incorporated herein by reference.

Cyclization to obtain the bicyclic guanidines can be performed usingthiocarbonyldiimidazole (CSIm₂) as shown in FIG. 1 and as described inthe ensuing Example. Alternatively, carbonyldiimidazole can be usedunder the same reaction conditions as those described forthiocarbonyldiimidazole in Example 1. Other reagents which can be usedto achieve cyclization to form the bicyclic guanidine include phosgene,triphosgene and thiophosgene. For example, bicyclic guanidines can beformed by treatment of the reduced acylated dipeptide with 24-foldexcess triphosgene for approximately fifteen minutes (0.1 M indichloromethane anhydrous with 5-fold excess of DIEA over dipeptide).The solution can then be removed, the resin washed with drydichloromethane for approximately twelve hours to let the cyclization goto completion. Similar procedures can be employed with thiophosgene ifan equimolar amount of non-nucleophilic base such asdiisopropylethylamine is added. Finally, as shown in FIG. 1, thecompounds can be cleaved from the resin using standard hydrogen fluorideprocedures.

Any variety of amino acids can be used with the present invention asdescribed above to generate a vast array of bicyclic guanidines withdifferent R¹ and R² groups. As described in the ensuing Example, fortynine different first Boc-protected amino acids were coupled to theresin, which amino acids contain R¹. The forty nine amino acids includedAla, Phe, Ile, Lys(Clz), Leu, Met(O), Arg(Tos), Val, Tyr(Brz), ala, phe,ile, lys(ClZ), leu, arg(Tos), val, tyr(Brz), α-Abu, α-Aib, Nva, nva,Nle, nle, Orn(Cbz), Nap, nap, Cha, cha, Met(O₂), pNO₂-Phe, pNO₂-phe,pCl-Phe, pCl-phe, pF-Phe, pf-phe, Lys(Ac), Pya, pya, Chg, chg, tBu-Gly,pNH₂-Phe(Fmoc), pNH₂-phe(Fmoc), Tyr(Et), tyr(Et), pI-Phe, pI-phe,Tyr(Me), and tyr(Me). Fifty one different second Boc-protected aminoacids were coupled, thereby providing fifty one various R²groups. Thosefifty one amino acids included Ala, Phe, Gly, Ile, Lys(Clz), Leu,Met(O), Arg(Tos), Val, Tyr(Brz), ala, phe, ile, lys(Clz), leu, arg(Tos),val, tyr(Brz), α-Abu, Nve, nve, Nle, nle, Orn(Cbz), Nap, nap, Cha, cha,Met(O₂), pNO₂-Phe, pNO₂-phe, pCl-Phe, pCl-phe, pF-Phe, pF-phe, Lys(Ac),Pya, pya, Chg, chg, tBu-Gly, pNH₂-Phe(Fmoc), pNH₂-phe(Fmoc), Tyr(Et),tyr(Et), Asp(Fm), asp(Fm), pI-Phe, pI-phe, Tyr(Me), and tyr(Me).

As used herein, abbreviations for the various amino acid side-chainprotecting groups are as follows: “tBu” for tert-butyl, “Boc” fortert-butoxycarbonyl, “Brz” for 2-bromobenzyloxycarbonyl, “Clz” for2-chlorobenzyloxycarbonyl, “Tos” for tosyl, “Cbz” for benzyloxycarbonyl,“Ac” for acetyl, “Fmoc” for fluorenylmethyloxycarbonyl, and “Fm” forfluorenylmethyl. These abbreviations and any others used herein arethose which are commonly known and used in the field. Moreover, also asis commonly practiced in the field and with reference to the amino acidnomenclature, all lower case lettering herein means the D-form of theamino acid as opposed to the L-form. Other nomenclature and three-letterabbreviations used herein for amino acids and derivatives thereof, aswell as their respective side chains are as follows:

TABLE 1 AMINO ACID NAME 3-LETTER SIDE CHAIN R FULL CODE (FOR R¹ AND R²)Alanine Ala —CH₃ Phenylalanine Phe

Glycine Gly —H Isoleucine Ile —CH(CH₃)CH₂CH₃ Lysine Lys —(CH₂)₄NH₂Leucine Leu —CH₂CH(CH₃)₂ Methionine- sulfoxide Met(O)

Methionine- sulfone Met(O₂)

Arginine Arg —CH₂CH₂CH₂NHC(NH)NH₂ Valine Val —CH(CH₃)₂ Tyrosine Tyr

O-Methyl-Tyrosine O-Me-Tyr or Tyr(Me)

O-Ethyl-Tyrosine O-Et-Tyr or Tyr(Et)

α-Aminobutyric α-Abu —CH₂—CH₃ acid α-Aminoisobutyric α-Aib —(CH₃)₂Norvaline Nva —CH₂CH₂CH₃ Norleucine Nle —CH₂CH₂CH₂CH₃ Ornithine Orn—(CH₂)₃NH₂ Napthylalanine Nap

Cyclohexylalanine Cha

p-nitro- Phenylalanine pNO₂-Phe

p-chloro- Phenylalanine pCl-Phe

p-fluoro- Phenylalanine pF-Phe

3-Pyridylalanine Pya

Cyclohexylglycine Chg

t-butyl-Glycine t-Bu-Gly —C(CH₃)3 p-amino- Phenylalanine pNH₂-Phe

p-iodo- Phenylalanine pI-Phe

Aspartic acid Asp —CH₂COOH

It should be appreciated from the above-described embodiments of R¹ andR², as well as from the described reaction scheme, that some of theamino acid side chains are modified during the synthesis. For instancesome of the R¹ amino acid side chains are modified by the reductionsteps. Similarly, certain R² groups are modified by the reductionprocedures. Accordingly, with reference to the forty nine preferredembodiments of R¹ and the fifty one of R², they are described above andbelow, except in Table 1, in their modified form. For example, followingreduction of a lysine side chain with a 2-chlorobenzyloxycarbonylprotecting group, an N-methylaminobutyl side chain would result.Following the guanidine formation step with thiocarbonyldiimidazole,this side chain would be further modified to form theN-methyl,N-thiocarbonylimidazole-aminobutyl functionality.

As well, a variety of carboxylic acids can be used in the acylation stepof Reaction Scheme I, thereby generating a wide array of substituents atthe R³ position of the bicyclic guanidines. Exemplary carboxylic acidsinclude the forty-one which were used in preparing the subjectcombinatorial libraries and compounds provided in the ensuing Example.Those forty one carboxylic acids include 3-phenylbutyric acid,m-toluylacetic acid, 3-fluorophenylacetic acid, p-toluylacetic acid,4-fluorophenylacetic acid, 3-methoxyphenylacetic acid,4-methoxyphenylacetic acid, 4-ethoxyphenylacetic acid,3-(3,4-dimethoxyphenyl)propionic acid, 4-biphenylacetic acid,(3,4-dimethoxyphenyl)acetic acid, phenylacetic acid, hydrocinnamic acid,4-phenylbutyric acid, butyric acid, heptanoic acid, isobutyric acid,(+/−)-2-methylbutyric acid, isovaleric acid, 3-methylvaleric acid,4-methylvaleric acid, (tert-butyl)acetic acid, cyclohexylcarboxylicacid, cyclohexylacetic acid, cyclohexylbutyric acid,cycloheptylcarboxylic acid, lactic acid, acetic acid,cyclobutylcarboxylic acid, cyclopentylcarboxylic acid,3-cyclopentylpropionic acid, cyclohexylpropionic acid,4-methyl-1-cyclohexylcarboxylic acid,4-(tert-butyl)-cyclohexylcarboxylic acid, 2-norbornylacetic acid,1-adamantaneacetic acid, 2-ethylbutyric acid, (3,3-diphenyl)propionicacid, 2-methyl-4-nitro-1-imidazolepropionic acid, cyclopentylaceticacid, and indolyl-3-acetic acid.

The bicyclic guanidine combinatorial library and compounds of Formula IIcan be prepared according to the general Reaction Scheme II in FIG. 2.The combinatorial libraries were prepared using solid-phase techniques.The solid-phase resin, here p-methylbenzhydrylamine resin (MBHA), isindicated in FIG. 2 by the circle and dash. Using traditional solidphase peptide chemistry, a first protected amino acid (having side chainR¹) is added to the resin. Following amino deprotection, a secondprotected amino acid (having side chain R²) is added and thendeprotected. A third protected amino acid (having side chain R³) is thenadded and deprotected. Exemplary amino acids are discussed in detailbelow.

The next step in the synthetic process, as shown in FIG. 2, is thereduction of the amide groups of the tripeptide using borane in THF togenerate three secondary amines. As discussed above, this method hasbeen used to generate diverse chemical libraries using the “librariesfrom libraries” concept as described, for instance, in Ostresh et al.and Cuervo et al., supra. When reduction is complete, the N-terminus isselectively protected by a triphenylmethyl group. The three remainingsecondary amines can be cyclized into a bicyclic guanidine usingthiocarbonyldiimadazole (CSIm₂), as shown in FIG. 2. Other cyclizingreagents such as thiophosgene can be used, as discussed above regardingReaction Scheme I.

The resulting positively charged resin-attached bicyclic guanidine canthen be washed and the group protecting the N-terminus removed with areagent such as 2% TFA. Following deprotection, the free N-terminus isacylated with one of a wide range of available carboxylic acid-derivedacyl groups to obtain the acylated tripeptide. Exemplary carboxylicacids are discussed in detail below. The resin then can be treated tolet the cyclization go to completion. Finally, as shown in FIG. 2, thecompounds can be cleaved from the resin using standard hydrogen fluorideprocedures.

Any variety of amino acids can be used with the present invention asdescribed above to generate a vast array of bicyclic guanidines withdifferent R¹, R² and R³ groups. As described in ensuing Example II,thirty-four different first Boc-protected amino acids were coupled tothe resin, which amino acids contain R¹. The thirty-four amino acidsincluded Ala, ala, Phe, phe, Ile, ile, Leu, leu, Val, val, Tyr(Brz),tyr(Brz), α-Abu, Aib, Nva, nva, Nle, nle, Nal, nal, Cha, cha, pF-Phe,pf-phe, pCl-Phe, pCl-phe, Chg, chg, Tyr(OEt), tyr(OEt), pI-Phe, pI-phe,Tyr(OMe), and tyr(OMe). Thirty-four different second Boc-protected aminoacids were coupled, thereby providing thirty-four various R²groups.Those thirty-four various amino acids included Ala, ala, Phe, phe, Ile,ile, Leu, leu, Val, val, Tyr(Brz), tyr(Brz), α-Abu, Nva, nva, Nle, nle,Nal, nal, Cha, cha, Met(O)₂, pF-Phe, pf-phe, pCl-Phe, pCl-phe, Chg, chg,Tyr(OEt), tyr(OEt), pI-Phe, pI-phe, Tyr(OMe), and tyr(OMe). Seventeendifferent third Boc-protected amino acids were coupled, therebyproviding seventeen various R³ groups. Those seventeen various aminoacids included Ala, ala, Phe, phe, Gly, Leu, leu, Nva, nva, Nle, nle,Cha, cha, Tyr(OEt), tyr(OEt), Tyr(OMe), and tyr(OMe). As describedabove, all lower case lettering herein means the D-form of the aminoacid as opposed to the L-form. Other nomenclature and three-letterabbreviations used herein for amino acids and derivatives thereof, aswell as their respective side chains, are described in Table 1 above.

As well, a variety of carboxylic acids can be used in the acylation stepof Reaction Scheme II, thereby generating a wide variety of substituentsat the R⁴ position of the bicyclic guanidines. Exemplary carboxylicacids that can be used as is or converted to the appropriate acylatingagent include the seventy-one which were used in preparing the subjectcombinatorial libraries and compounds provided in Example II below.Those seventy-one carboxylic acids include 1-phenyl-1-cyclopropanecarboxylic acid, 2-phenylbutyric acid, 3-phenylbutyric acid,m-toluylacetic acid, 3-fluorophenylacetic acid, 3-bromophenylaceticacid, (α,α,α-trifluoro-m-toluyl)acetic acid, p-toluylacetic acid,3-methoxyphenylacetic acid, 4-bromophenylacetic acid,4-methoxyphenylacetic acid, 4-ethoxyphenylacetic acid,4-isobutyl-α-methylphenylacetic acid, 3,4-dichlorophenylacetic acid,3-(3,4-dimethoxyphenyl)propionic acid, 4-biphenylacetic acid,α-methylcinnamic acid, 2-(trifluoromethyl)cinnamic acid,(3,4-dimethoxyphenyl)acetic acid, 3,4-(methylenedioxy)phenylacetic acid,2-methoxycinnamic acid, benzoic acid, 4-chlorocinnamic acid, m-anisicacid, 4-isopropylbenzoic acid, 4-vinylbenzoic acid, 4-fluorobenzoicacid, 4-bromobenzoic acid, 3,4-dimethoxycinnamic acid, t-cinnamic acid,3,4-dimethylbenzoic acid, 3-fluoro-4-methylbenzoic acid,3-bromo-4-methylbenzoic acid, 3-iodo-4-methylbenzoic acid,3,4-dichlorobenzoic acid, 4-biphenylcarboxylic acid, 3,4-difluorobenzoicacid, m-toluic acid, phenylacetic acid, hydrocinnamic acid,3-methoxy-4-methylbenzoic acid, 4-phenylbutyric acid,3,4-dimethoxybenzoic acid, 4-ethyl-4-biphenylcarboxylic acid,3,4,5-trimethoxybenzoic acid, butyric acid, heptanoic acid, isobutyricacid, (+/−)-2-methylbutyric acid, isovaleric acid, 3-methylvaleric acid,4-methylvaleric acid, p-toluic acid, p-anisic acid, cyclohexylcarboxylicacid, cyclohexylacetic acid, cyclohexylbutyric acid,cycloheptylcarboxylic acid, acetic acid, 2-methylcyclopropylcarboxylicacid, cyclobutylcarboxylic acid, cyclopentylcarboxylic acid,3-cyclopentylpropionic acid, 2-furoic acid, cyclohexylpropionic acid,4-methyl-1-cyclohexylcarboxylic acid, 4-t-butylcyclohexylcarboxylicacid, 4-methylcyclohexylacetic acid, tiglic acid, 2-norbornylaceticacid, and 2-thiophenecarboxylic acid.

The nonsupport-bound combinatorial library mixtures were screened insolution in radio-receptor inhibition assays and in an anti-bacterialassay, an anti-fungal assay, a calmodulin-dependent phosphodiesterase(CaMPDE) assay and a phosphodiesterase (PDE) assay described in detailbelow. Deconvolution of highly active mixtures can then be carried outby iterative or positional scanning methods. These techniques, theiterative approach or the positional scanning approach, can be utilizedfor finding other active compounds within the combinatorial libraries ofthe present invention using any one of the below-described assays orothers well known in the art.

The iterative approach is well-known and is set forth in general inHoughten et al., Nature, 354, 84-86 (1991) and Dooley et al., Science,266, 2019-2022 (1994), both of which are incorporated herein byreference. In the iterative approach, for example, sub-libraries of amolecule having three variable groups are made wherein the firstvariable is defined. Each of the compounds with the defined variablegroup is reacted with all of the other possibilities at the other twovariable groups. These sub-libraries are each tested to define theidentity of the second variable in the sub-library having the highestactivity in the screen of choice. A new sub-library with the first twovariable positions defined is reacted again with all the otherpossibilities at the remaining undefined variable position. As before,the identity of the third variable position in the sub-library havingthe highest activity is determined. If more variables exist, thisprocess is repeated for all variables, yielding the compound with eachvariable contributing to the highest desired activity in the screeningprocess. Promising compounds from this process can then be synthesizedon larger scale in traditional single-compound synthetic methods forfurther biological investigation.

The positional-scanning approach has been described for variouscombinatorial libraries as described, for example, in R. Houghten et al.PCT/US91/08694 and U.S. Pat. No. 5,556,762, both of which areincorporated herein by reference. The positional scanning approach isused as described below in the preparation and screening of thecombinatorial libraries. In the positional scanning approachsublibraries are made defining only one variable with each set ofsublibraries and all possible sublibraries with each single variabledefined (and all other possibilities at all of the other variablepositions), made and tested. From the instant description one skilled inthe art could synthesize combinatorial libraries wherein two fixedpositions are defined at a time. From the testing of eachsingle-variable defined combinatorial library, the optimum substituentat that position is determined, pointing to the optimum or at least aseries of compounds having a maximum of the desired biological activity.Thus, the number of sublibraries for compounds with a single positiondefined will be the number of different substituents desired at thatposition, and the number of all the compounds in each sublibrary will bethe product of the number of substituents at each of the othervariables.

Individual compounds and pharmaceutical compositions containing the newbicyclic guanidines, as well as methods of using the same, are includedwithin the scope of the present invention. The new bicyclic guanidinecompounds of the present invention can be used for a variety of purposesand indications and as medicaments for any such purposes andindications. For example, guanidine moieties are found in manybiologically active compounds and, as described above, can be used toblock hypotensive and adrenergic effects, E. J. Corey and MitsuakiOhtani, Tetrahedron Letters., 30(39):5227-5230 (1989), incorporatedherein by reference, or as sweeteners Nagarajan et al. SyntheticCommunications., 22(8):1191-1198 (1992), incorporated herein byreference. Additionally, as shown by the present invention, the subjectcompounds are useful as analgesics. Assays which can be, some of whichhave been, used to test the biological activity of the instant bicyclicguanidines include antimicrobial assays, a competitive enzyme-linkedimmunoabsorbent assay and radio-receptor assays, as described below andwhose results are shown in Examples IV, V and VI.

The ability of the compounds to inhibit bacterial growth, and thereforebe useful to that infection, can be determined by methods well known inthe art. An exemplary in vitro antimicrobial activity assay is describedin Blondelle and Houghten, Biochemistry 30:4671-4678 (1991), which isincorporated herein by reference. In brief, Staphylococcus aureus ATCC29213 (Rockville, Md.) is grown overnight at 37° C. in Mueller-Hintonbroth, then re-inoculated and incubated at 37° C. to reach theexponential phase of bacterial growth (i.e., a final bacterialsuspension containing 10⁵ to 5×10⁵ colony-forming units/ml). Theconcentration of cells is established by plating 100 μl of the culturesolution using serial dilutions (e.g., 10⁻², 10⁻³ and 10⁻⁴) onto solidagar plates. In 96-well tissue culture plates bicyclic guanidines,individual or in mixtures, are added to the bacterial suspension atconcentrations derived from serial two-fold dilutions ranging from 1500to 2.9 μg/ml. The plates are incubated overnight at 37° C. and thegrowth determined at each concentration by OD₆₂₀ nm. The IC₅₀ (theconcentration necessary to inhibit 50% of the growth of the bacteria)can then be calculated.

The competitive ELISA method which can be used here is a modification ofthe direct ELISA technique described previously in Appel et al., J.Immunol. 144:976-983 (1990), which is incorporated herein by reference.It differs only in the MAb addition step. Briefly, multi-wellmicroplates are coated with the antigenic peptide (Ac-GASPYPNLSNQQT-NH₂)at a concentration of 100 pmol/50 μl. After blocking, 25 μl of a 1.0mg/ml solution of each bicyclic guanidine mixture of a syntheticcombinatorial library (or individual bicyclic guanidine) is added,followed by MAb 125-10F3 (Appel et al., supra) (25 μl per well). The MAbis added at a fixed dilution in which the bicyclic guanidine in solutioneffectively competes for MAb binding with the antigenic peptide adsorbedto the plate. The remaining steps are the same as for direct ELISA. Theconcentration of bicyclic guanidine necessary to inhibit 50% of the MAbbinding to the control peptide on the plate (IC₅₀) is determined byserial dilutions of the bicyclic guanidine.

Alternative screening can be, and has been, done with radio-receptorassays as provided in Examples IV, V and VI and FIGS. 3 and 4. Theradio-receptor assay, can be selective for any one of the μ, κ, or δopiate receptors. Therefore, the compounds of the present invention areuseful in vitro for the diagnosis of relevant opioid receptor subtypes,such as κ, in the brain and other tissue samples. Similarly, thecompounds can be used in vivo diagnostically to localize opioid receptorsubtypes.

The radio-receptor assays are also an indication of the compounds'analgesic properties as described, for example, in Dooley et al., Proc.Natl. Acad. Sci., 90:10811-10815 (1993). For example, it can beenvisioned that these compounds can be used for therapeutic purposes toblock the peripheral effects of a centrally acting pain killer. Forinstance, morphine is a centrally acting pain killer. Morphine, however,has a number of deleterious effects in the periphery which are notrequired for the desired analgesic effects, such as constipation andpruritus (itching). While it is known that the many compounds do notreadily cross the blood-brain barrier and, therefore, elicit no centraleffect, the subject compounds can have value in blocking the peripheryeffects of morphine, such as constipation and pruritus. Accordingly, thesubject compounds are also useful as drugs, namely as analgesics, or totreat pathologies associated with other compounds which interact withthe opioid receptor system.

Additionally, such compounds can be tested in a σ receptor assay.Ligands for the σ receptor can be useful as antipsychotic agents, asdescribed in Abou-Gharbia et al., Annual Reports in Medicinal Chemistry,28:1-10 (1993).

Radio-receptor assays, such as those whose results are shown in ExamplesIV, V and VI, below, can be performed with particulate membranesprepared using a modification of the method described in Pasternak etal., Mol. Pharmacol. 11:340-351 (1975), which is incorporated herein byreference. Rat brains frozen in liquid nitrogen can be obtained fromRockland (Gilbertsville, Pa.). The brains are thawed, the cerebellaremoved and the remaining tissue weighed. Each brain is individuallyhomogenized in 40 ml Tris-HCl buffer (50 mM, pH 7.4, 4° C.) andcentrifuged (Sorvall® RC5C SA-600: Du Pont, Wilmington, Del.) (16,000rpm) for 10 minutes. The pellets are resuspended in fresh Tris-HClbuffer and incubated at 37° C. for 40 minutes. Following incubation, thesuspensions are centrifuged as before, the resulting pellets resuspendedin 100 volumes of Tris buffer and the suspensions combined. Membranesuspensions are prepared and used in the same day. Protein content ofthe crude homogenates generally range from 0.15-0.2 mg/ml as determinedusing the method described in Bradford, M. M., Anal. Biochem. 72:248-254(1976), which is incorporated herein by reference.

Binding assays are carried out in polypropylene tubes, each tubecontaining 0.5 ml of membrane suspension. 8 nM of³H-[D-Ala²,Me-Phe⁴,Gly-ol⁵]enkephalin (DAMGO) (specific activity=36Ci/mmol, 160,000 cpm per tube; which can be obtained from MultiplePeptide Systems, San Diego, Calif., through NIDA drug distributionprogram 271-90-7302) and 80 μg/ml of bicyclic guanidine, individual oras a mixture and Tris-HCl buffer in a total volume of 0.65 ml. Assaytubes are incubated for 60 mins. at 25° C. The reaction is terminated byfiltration through GF-B filters on a Tomtec harvester (Orange, Conn.).The filters are subsequently washed with 6 ml of Tris-HCl buffer, 4° C.Bound radioactivity is counted on a Pharmacia Biotech Betaplate LiquidScintillation Counter (Piscataway, N.J.) and expressed in cpm. Todetermine inter- and intra-assay variation, standard curves in which³H-DAMGO is incubated in the presence of a range of concentrations ofunlabeled DAMGO (0.13-3900 nM) are generally included in each plate ofeach assay (a 96-well format). Competitive inhibition assays areperformed as above using serial dilutions of the bicyclic guanidines,individually or in mixtures. IC₅₀ values (the concentration necessary toinhibit 50% of ³H-DAMGO binding) are then calculated. IC₅₀ values ofless than 1000 nM are indicative of highly active opioid compounds whichbind to the μ receptor, with particularly active compounds having IC₅₀values of 100 nM or less and the most active compounds with values ofless than 10 nM.

As opposed to this μ receptor selective assay, which can be carried outusing ³H-DAMGO as radioligand, as described above, assays selective forκ receptors can be carried out using [³H]-U69,593 (3 nM, specificactivity 62 Ci/mmol) as radioligand. Assays selective for δ opiatereceptors can be carried out using tritiated DSLET ([D-Ser²,D-Leu⁵]-threonine-enkephalin) as radioligand. Assays selective for the σopiate receptor can use radiolabeled pentazocine as ligand.

Screening of combinatorial libraries and compounds of the invention alsocan be, and has been, done with an anti-fungal assay as provided inExamples VII and VIII and FIG. 5. Many compounds were shown to beactive. Therefore, compounds of the present invention are useful fortreating fungal infections.

Fungal infections, including life threatening infections cause bypathogenic fungi, are becoming increasingly common, especially in thoseindividuals with suppressed immune systems such as those with cancer orAIDS. In particular, Candida albicans and Cryptococcus neoformans aretwo of the most common fungi responsible for infections. Candidiasis isthe fungal infection most frequently associated with HIV-positivepatients. Cryptococcosis is the leading cause of morbidity and mortalitydue to fungi in those with AIDS. The compounds of the subject inventionare useful for treating these, as well as other, fungal infections.

An example of an anti-fungal assay is the one whose results are shown inExamples VII and VIII, below. Microdilution assays can be carried outagainst Candida albicans (ATCC 10231) in ninety-six-well tissue cultureplates, as described in Blondelle et al., J. Appl. Bacteriol., 78:39(1995). In brief, the yeast culture are spread on YM agar plates andincubated at 30° C. for 48 hours prior to the assay. Two colonies ofthis culture (each 1 mm in diameter) are then seeded in 5 ml of 2×YMbroth, vortex-mixed and diluted 10-fold in 2×YM broth, for anapproximate final concentration of 10⁵ to 5×10⁵ CFU/ml. The actualCandida albicans concentration are determined by plating on agar platesas described above. Yeast suspension in 2×broth are added to themixtures at varying concentrations derived from serial two-folddilutions. The plates are then incubated for 48 hours at 30° C. Therelative percent growth of the yeast found for each mixture can bedetermined by the optical density at 620 nm (OD₆₂₀) using a TitertekMultiskan Plus apparatus. The IC₅₀ values then can be calculated using asigmoidal curve fitting software (Graphpad, ISI Software, San Diego,Calif.). The minimum inhibitory concentration (MIC), which is defined asthe lowest concentration of mixture at which no change in OD₆₂₀ occursbetween 0 and 48 hours, also can be determined.

A bicyclic guanidine synthetic combinatorial library was assayed inpositional scanning format for antifungal activity against Candidaalbicans. Each mixture was screened at four concentrations varying from250 to 31.25 μg/ml or, for the most active mixtures, at eightconcentrations varying from 250 to 1.95 μg/ml and their IC₅₀ valuesdetermined (FIG. 5; and Table 12, Example VII). Following the screening,thirty-two individual compounds were synthesized and screened in asimilar manner. The most active compounds showed MIC values of 3-8 μg/ml(Table 13, Example VIII).

Screening of combinatorial libraries and compounds of the invention alsocan be, and has been, done with a calmodulin-dependent phosphodiesterase(CaMPDE) assay as provided in Examples IX to XII and FIG. 6. Manycompounds were shown to be active. Therefore, compounds of the presentinvention are useful as calmodulin antagonists.

Calmodulin (CaM), which is the major intracellular calcium receptor, isinvolved in many processes that are crucial to cellular viability. Inparticular, calmodulin is implicated in calcium-stimulated cellproliferation. Calmodulin antagonists are, therefore, useful fortreating conditions associated with increased cell proliferation, forexample, cancer. In addition, calmodulin antagonists such as compoundsof the subject invention are useful both in vitro and in vivo foridentifying the role of calmodulin in other biological processes. Thedisadvantages of known antagonists such as trifluoperazine andN-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W13) include theirnon-specificity and toxicity. In contrast, advantages of the cycliccombinatorial libraries and compounds of the subject invention ascalmodulin antagonists include their reduced flexibility and ability togenerate broader conformational space of interactive residues ascompared to their linear counterparts.

An example of an assay that identifies CaM antagonists is a CaMPDEassay, such as the one whose results are shown in Examples IX to XII,below. Samples are mixed with 50 μl of assay buffer (360 mM Tris, 360 mMImidazole, 45 mM Mg(CH₃COO)₂, pH 7.5) and 10 μl of CaCl₂ (4.5 mM) to afinal volume of 251 μl. 25 μl of calmodulin stock solution (BoehringerMannheim; 0.01 μg/μl) is then added and the samples then sit at roomtemperature for 10 minutes. 14 μl of PDE (Sigma; 2 Units dissolved in 4ml of water; stock concentration: 0.0005 Units/μl) is then added,followed by 50 μl of 5′-nucleotidase (Sigma; 100 Units dissolved in 10ml of 10 mM Tris-HCl containing 0.5 mM Mg(CH₃COO)₂, pH 7.0; stockconcentration: 10 Units/ml). The samples are then incubated for 10minutes at 30° C. 50 μl of adenosine 3′,5′-cyclic monophosphate (cAMP)(20 mM in water at pH 7.0) is added, the samples incubated for 1 hour at30° C. and then vortexed. 200 μl of trichloroacetic acid (TCA) (55% inwater) is added to a 200 μl sample aliquot, which is then vortexed andcentrifuged for 10 minutes. 80 μl of the resulting supernatants of eachsample is transferred to a 96-well plate, with 2 wells each containing80 μl of each sample. 80 μl of ammonium molybdate (1.1% in 1.1N H₂SO₄)is then added to all the wells, and the OD of each were determined at730 nm, with the values later subtracted to the final OD reading. 16 μlof reducing agent (6 g sodium bisulfite, 0.6 g sodium sulfite and 125 mgof 1-amino-2-naphtol-4-sulfonic acid in 50 ml of water) is then added toone of each sample duplicate and 16 μl of water is added to the otherduplicate. After sitting for 1 hour at room temperature, the OD of eachwell is determined at 730 nm. The percent inhibition of calmodulinactivity is then calculated for each sample, using as 0% inhibition acontrol sample containing all reagents without any test samples and as100% inhibition a control sample containing test samples and allreagents except calmodulin. In addition, the percent inhibition ofphosphodiesterase activity was determined by following a similarprotocol as the CaMPDE assay described above, except not addingcalmodulin to the sample mixture and calculating the percent inhibitionby using as 0% inhibition a control reagent without any test samples andas 100% inhibition a control sample containing test samples and allreagents except cAMP.

A bicyclic guanidine synthetic combinatorial library was assayed inpositional scanning format for activity as calmodulin antagonists, asdescribed above. Each mixture contained 2,000 to 2,500 individualcompounds. At 15 μl/mg, over half of the mixtures showed greater than60% inhibition (FIG. 6; Table 14 of Example IX). The IC₅₀ values werethen determined for the most active mixtures, which were screened atfour different concentrations varying from 50 to 2 μl/mg (Table 15 ofExample X).

Since a large number of known calmodulin antagonists are not specific tocalmodulin but also interact with target enzymes, these active mixtureswere also assayed for specificity toward calmodulin versusphosphodiesterase (Table 16 of Example XI). Based on this screeningdata, individual bicyclic guanidine compounds were assayed forinhibitory activities in a similar manner as the mixtures, withdilutions varying from 10 to 1 μl/mg. The IC₅₀ values of theseindividual compounds ranged from about 0.8 to about 12 μl/ml, whichrepresents a 10 to 20-fold increase compared to known antagonists suchas trifluoperazine.

The novel compounds of the subject invention can be incorporated intopharmaceutical compositions. As pharmaceutical compositions foreffecting analgesia, treating infections, pain, or other indicationsknown to be treatable by guanidines, the bicyclic guanidine compounds ofthe present invention are generally in a pharmaceutical composition soas to be administered to a subject at dosage levels of from 0.7 to 7000mg per day, and preferably 1 to 500 mg per day, for a normal human adultof approximately 70 kg of body weight, this translates into a dosage offrom 0.01 to 100 mg/kg of body weight per day. The specific dosagesemployed, however, can be varied depending upon the requirements of thepatient, the severity of the condition being treated, and the activityof the compound being employed. The determination of optimum dosages fora particular situation is within the skill of the art.

For preparing pharmaceutical compositions containing compounds of theinvention, inert, pharmaceutically acceptable carriers are used. Thepharmaceutical carrier can be either solid or liquid. Solid formpreparations include, for example, powders, tablets, dispersiblegranules, capsules, cachets, and suppositories.

A solid carrier can be one or more substances which can also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, or tablet disintegrating agents; it can also be anencapsulating material.

In powders, the carrier is generally a finely divided solid which is ina mixture with the finely divided active component. In tablets, theactive compound is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

For preparing pharmaceutical composition in the form of suppositories, alow-melting wax such as a mixture of fatty acid glycerides and cocoabutter is first melted and the active ingredient is dispersed thereinby, for example, stirring. The molten homogeneous mixture is then pouredinto convenient-sized molds and allowed to cool and solidify.

Powders and tablets preferably contain between about 5% to about 70% byweight of the active ingredient. Suitable carriers include, for example,magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin,dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethylcellulose, a low-melting wax, cocoa butter and the like.

The pharmaceutical compositions can include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component (with or without other carriers)is surrounded by a carrier, which is thus in association with it. In asimilar manner, cachets are also included. Tablets, powders, cachets,and capsules can be used as solid dosage forms suitable for oraladministration.

Liquid pharmaceutical compositions include, for example, solutionssuitable for oral or parenteral administration, or suspensions, andemulsions suitable for oral administration. Sterile water solutions ofthe active component or sterile solutions of the active component insolvents comprising water, ethanol, or propylene glycol are examples ofliquid compositions suitable for parenteral administration.

Sterile solutions can be prepared by dissolving the active component inthe desired solvent system, and then passing the resulting solutionthrough a membrane filter to sterilize it or, alternatively, bydissolving the sterile compound in a previously sterilized solvent understerile conditions.

Aqueous solutions for oral administration can be prepared by dissolvingthe active compound in water and adding suitable flavorants, coloringagents, stabilizers, and thickening agents as desired. Aqueoussuspensions for oral use can be made by dispersing the finely dividedactive component in water together with a viscous material such asnatural or synthetic gums, resins, methyl cellulose, sodiumcarboxymethyl cellulose, and other suspending agents known to thepharmaceutical formulation art.

Preferably, the pharmaceutical composition is in unit dosage form. Insuch form, the composition is divided into unit doses containingappropriate quantities of the active bicyclic guanidine. The unit dosageform can be a packaged preparation, the package containing discretequantities of the preparation, for example, packeted tablets, capsules,and powders in vials or ampules. The unit dosage form can also be acapsule, cachet, or tablet itself, or it can be the appropriate numberof any of these packaged forms.

The following Examples are intended to illustrate but not limit thepresent invention.

Introduction

When using either the iterative or positional scanning approach to thesynthesis of the instant combinatorial libraries, it is necessary atsome point to expose either the solid phase alone or the solid phasebound to one or two amino acids, to a mixture of reactive subunits. Suchsubunits can be the first or second amino acid, or the activatedcarboxylic acid residue. As each individual subunit in the mixture mayreact at varying rates with the solid phase or the molecule bound to thesolid phase, it is advantageous to know the relative reaction rate ofeach reactive subunit. Once such relative rates are known, theconcentration of each reactive subunit can be adjusted accordingly inorder to have approximately equimolar amounts of each reactive subunitcouple with either the bare solid support or the molecule bound to thesupport. (For a further discussion of this point, see J. M. Ostresh etal., Biopolymers, 34:1661-1689 (1994), herein incorporated byreference).

The theory underpinning the methodology for determining the relativereaction rates used by Ostresh et al. in the above-mentioned Biopolymersarticle is set forth below.

Assuming that a large excess of the amino acid to be reacted with thePeptide which in turn is bound to the solid support is used, then therate of such a reaction for amino acid 1 and amino acid 2 is expressedin Equations (1) and (2) below, respectively:

(Peptide−AA₁)=k_(AA-1)×(AA₁)  (1)

wherein:

“Peptide”=Ala-Phe-Leu-;

AA₁=baseline amino acid; and

k_(AA-1)=reaction constant of AA₁ with Peptide.

(Peptide−AA₂)=k_(AA-2)×(AA₂)  (2)

wherein:

“Peptide”=Ala-Phe-Leu-;

AA₂=amino acid whose reaction rate with Peptide is to be compared toAA₁; and

k_(AA-2)=reaction rate of AA₂ with the Peptide.

If k_(AA-1) and k_(AA-2) are different, then for any given period oftime, more of the AA with the slower rate must be added to the mixtureof reactive subunits so that the Peptide attached to the solid supportwill have reacted at that step with approximately equal amounts of AA₁and AA₂. Thus, only relative rates are of importance, and can bedetermined using the following equations: $\begin{matrix}\frac{k_{{AA} - 1} = {\left( {{peptide} - {AA}_{1}} \right) \times \left( {AA}_{2} \right)}}{k_{{AA} - 2} = {\left( {{peptide} - {AA}_{2}} \right) \times \left( {AA}_{1} \right)}} & (3)\end{matrix}$

In order to simplify the calculations, a ten fold molar excess of bothAA₁ and AA₂ are used in experiments coupling the AA in question to thesolid support “Peptide”; allowing Equation 3 to be simplified toEquation 4: $\begin{matrix}{\frac{k_{{AA} - 1}}{k_{{AA} - 2}} = \frac{\left( {{peptide} - {AA}_{1}} \right)}{\left( {{peptide} - {AA}_{2}} \right)}} & (4)\end{matrix}$

on the assumption that (AA₁)=(AA₂).

In order to determine the proper ratio of concentrations of AA₁ and AA₂to use in a reaction mixture; Equations (3) and (4) are solved for [AA₁]and [AA₂] to give Equation (5): $\begin{matrix}{\frac{\left( {AA}_{2} \right)}{\left( {AA}_{1} \right)} = \frac{k_{{AA} - 1}\left( {{peptide} - {AA}_{2}} \right)}{k_{{AA} - 2}\left( {{peptide} - {AA}_{1}} \right)}} & (5)\end{matrix}$

Since equimolar concentrations of Peptide-AA₁ and Peptide-AA₂ aredesired equation (5) simplifies to Equation (6): $\begin{matrix}{\frac{\left( {AA}_{2} \right)}{\left( {AA}_{1} \right)} = \frac{k_{{AA} - 1}}{k_{{AA} - 2}}} & (6)\end{matrix}$

The ratio in Equation (6) was determined using the followingmodification of the Biopolymers article procedure. Thus, instead ofcleaving and hydrolysing the peptide with 6N hydrochloric acid,equimolar amounts of Peptide-AA₁ and Peptide-AA₂, each bound separatelyto the same type of solid support used in the reactions of AA₁ and AA₂,were mixed with the reaction mixtures. The peptides were then cleavedand analyzed by HPLC (5-65% B in 30 minutes, Vydac 218TP54, A:0.05%TFA/H₂O, B:0.05% TFA/ACN, 214 nm). Carboxylic acid ratios were generatedusing the same technique.

The relative ratios for the reactive amino acid and carboxylic acidsubunits determined by the above methodology are set forth,respectively, in Tables 2 and 3 below. These ratios apply regardless ofto which structure (Formula I or Formula II) or at which position thesubunit is being added.

TABLE 2 Predetermined Ratios No. Amino Acid Ratio  1 Boc-L-Alanine 0.95 2 Boc-L-Phenylalanine 0.81  3 Boc-L-Isoleucine 1.16  4 Boc-L-Lysine(2-ClZ) 1.05  5 Boc-L-Leucine 1.08  6 Boc-L-Methionine (O) 0.89  7Boc-L-Arginine (Tos) 1.42  8 Boc-L-Valine 1.14  9 Boc-L-Tyrosine(2-Brz)1.26 10 Boc-D-alanine 0.95 11 Boc-D-phenylalanine 0.81 12Boc-D-isoleucine 1.16 13 Boc-D-lysine (Clz) 1.05 14 Boc-D-leucine 1.0815 Boc-D-arginine (Tos) 1.42 16 Boc-D-valine 1.14 17 Boc-D-tyrosine(Brz) 1.26 18 Boc-L-α-Aminobutyric acid 0.94 19 Boc-α-Aminoisobutyricacid 1.66 20 Boc-L-Norvaline 1.15 21 Boc-D-norvaline 1.15 22Boc-L-norleucine 1.15 23 Boc-D-norleucine 1.15 24 Boc-L-Ornithine (Cbz)1.06 25 Boc-L-Naphthylalanine 0.55 26 Boc-D-naphthylalanine 0.55 27Boc-L-cyclohexylalanine 1.50 28 Boc-D-cyclohexylalanine 1.50 29Boc-L-Methionine sulfone 0.90 30 Boc-L-p-nitro-Phenylalanine 1.00 31Boc-D-p-nitro-phenylalanine 1.00 32 Boc-L-p-chloro-Phenylalanine 1.00 33Boc-D-p-chloro-phenylalanine 1.00 34 Boc-L-p-fluoro-Phenylalanine 1.0035 Boc-D-p-fluoro-phenylalanine 1.00 36 Boc-L-Lysine (Ac) 0.90 37Boc-L-(3-Pyridyl)alanine 1.00 38 Boc-D-(3-pyridyl)alanine 1.00 39Boc-L-Cyclohexylglycine 2.00 40 Boc-D-cyclohexylglycine 2.00 41Boc-L-α-tButylglycine 2.00 42 Boc-p-Fmoc-amino-L-Phenylalanine 1.25 43Boc-p-Fmoc-amino-D-phenylalanine 1.25 44 Boc-O-Ethyl-L-Tyrosine 1.20 45Boc-O-Ethyl-D-tyrosine 1.20 46 Boc-p-Iodo-L-Phenylalanine 1.00 47Boc-p-Iodo-D-phenylalanine 1.00 48 Boc-O-Methyl-L-Tyrosine 1.20 49Boc-O-Methyl-D-tyrosine 1.20 50 Boc-Glycine 1.00 51 Boc-L-Aspartic acid(Fm) 1.00 52 Boc-D-aspartic acid (Fm) 1.00

TABLE 3 Predetermined Ratios No. Carboxylic Acid Ratio  13-phenylbutyric acid 2.6  2 m-toluylacetic acid 1.8  33-fluorophenylacetic acid 0.84  4 p-toluylacetic acid 1.36  54-fluorophenylacetic acid 1.04  6 3-methoxyphenylacetic acid 1.17  74-methoxyphenylacetic acid 1.8  8 4-ethoxyphenylacetic acid 1.4  93-(3,4-dimethoxyphenyl)-propionic 2.2 acid 10 4-biphenylacetic acid 1.411 3,4-dimethoxyphenylacetic acid 1.44 12 phenylacetic acid 1 13hydrocinnamic acid 2.5 14 4-phenylbutyric acid 3 15 butyric acid 3.4 16heptanoic acid 3.51 17 isobutyric acid 3.11 18 2-methylbutyric acid 6.2519 isovaleric acid 6.36 20 3-methylvaleric acid 5.06 21 4-methylvalericacid 3.32 22 (tert-butyl)acetic acid 6 23 cyclohexylcarboxylic acid 3.5124 cyclohexylacetic acid 3.95 25 cyclohexylbutyric acid 3.33 26cycloheptanecarboxylic acid 2.6 27 lactic acid 0.25 28 acetic acid 2.6529 cyclobutanecarboxylic acid 2.77 30 cyclopentanecarboxylic acid 3.0331 3-cyclopentylpropionic acid 3.71 32 cychexylpropionic acid 2.8 334-methyl-1-cyclohexylcarboxylic acid 5.92 344-(tert-butyl)-cyclohexylcarboxylic 6.64 acid 35 2-norbornylacetic acid5.45 36 1-adamantaneacetic acid 11.16 37 2-ethylbutyric acid 6 383,3-diphenylpropionic acid 2.8 39 2-methyl-4-nitro-1-imidazolepropionic0.81 acid 40 cyclopentylacetic acid 3.96 41 indole-3-acetic acid 3 421-phenyl-1-cyclopropane carboxylic 1.00 acid 43 2-phenylbutyric acid1.20 44 3-bromophenylacetic acid 0.61 45 (α,α,α-trifluoro-m-toluyl)acetic 0.61 acid 46 4-isobutyl-α-methylphenylacetic acid 1.70 473,4-dichlorophenylacetic acid 0.81 48 α-methylcinnamic acid 1.95 492-(trifluoromethyl)cinnamic acid 1.03 503,4-(methylenedioxy)-phenylacetic 1.27 acid 51 2-methoxycinnamic acid5.60 52 benzoic acid 1.28 53 4-chlorocinnamic acid 2.95 54 m-anisic acid1.52 55 4-isopropylbenzoic acid 3.00 56 4-vinylbenzoic acid 1.50 574-flourobenzoic acid 1.22 58 4-bromobenzoic acid 0.59 593,4-dimethoxycinnamic acid 7.27 60 t-cinnamic acid 4.20 613,4-dimethylbenzoic acid 2.44 62 3-fluoro-4-methylbenzoic acid 0.75 633-bromo-4-methylbenzoic acid 0.86 64 3-iodo-4-methylbenzoic acid 0.64 653,4-dichlorobenzoic acid 0.39 66 4-biphenylcarboxylic 5.10 673,4-difluorobenzoic acid 0.45 68 m-toluic acid 1.60 693-methoxy-4-methylbenzoic acid 2.10 70 3,4-dimethoxybenzoic acid 3.08 714-ethyl-4-biphenylcarboxylic acid 0.92 72 3,4,5-trimethoxybenzoic acid1.46 73 p-toluic acid 2.28 74 p-anisic acid 5.38 752-methylcyclopropylcarboxylic acid 2.42 76 2-furoic acid 4.44 774-methylcyclohexylacetic acid 4.79 78 tiglic acid 4.59 792-thiophenecarboxylic acid 1.16

EXAMPLE I

This example provides the synthesis of a combinatorial library of thepresent invention according to Reaction Scheme I, which is shown in FIG.1. The R¹, R² and R³ groups varied as described above and below. Again,forty-nine first amino acids were used, generating at least forty-nineR¹ groups, depending on the modifications to the side chains. The aminoacids used to generate the R¹ groups are again listed below in Table 4.Fifty-one second amino acids were used to generate the various R²groups, which amino acids are also again summarized in Table 4 below.Finally, the forty-one carboxylic acids used to acylate the dipeptidesand generate the various R³ groups are also listed again in Table 4.Therefore, Table 4 provides a summary of all the amino acids (R¹ and R²)and carboxylic acid components (R³) used in the preparation of thecombinatorial library.

TABLE 4 SUMMARY OF REAGENTS USED TO GENERATE R GROUPS IN PREPAREDLIBRARIES R¹ R² R³  1 Ala Ala 3-phenylbutyric acid  2 Phe Phem-toluylacetic acid  3 Ile Gly 3-fluorophenylacetic acid  4 Lys(Clz) Ilep-toluylacetic acid  5 Leu Lys(Clz) 4-fluorophenylacetic acid  6 Met(O)Leu 3-methoxyphenylacetic acid  7 Arg(Tos) Met(O) 4-methoxyphenylaceticacid  8 Val Arg(Tos) 4-ethoxyphenylacetic acid  9 Tyr(Brz) Val3-(3,4-dimethoxyphenyl)- propionic acid 10 ala* Tyr(Brz)4-biphenylacetic acid 11 phe ala* (3,4-dimethoxyphenyl)acetic acid 12ile phe phenylacetic acid 13 lys(Clz) ile hydrocinnamic acid 14 leulys(Clz) 4-phenylbutyric acid 15 arg(Tos) leu butyric acid 16 valarg(Tos) heptanoic acid 17 tyr(Brz) val isobutyric acid 18 α-Abutyr(Brz) (+/−)-2-methylbutyric acid 19 α-Aib α-Abu isovaleric acid 20Nve Nve 3-methylvaleric acid 21 nve nve 4-methylvaleric acid 22 Nle Nle(tert-butyl)acetic acid 23 nle nle cyclohexylcarboxylic acid 24 Orn(Cbz)Orn(Cbz) cyclohexylacetic acid 25 Nap Nap cyclohexylbutyric acid 26 napnap cycloheptanecarboxylic acid 27 Cha Cha lactic acid 28 cha cha aceticacid 29 Met(O₂) Met(O₂) cyclobutanecarboxylic acid 30 pNO₂-Phe pNO₂-Phecyclopentanecarboxylic acid 31 pNO₂-phe pNO₂-phe 3-cyclopentylpropionicacid 32 pCl-Phe pCl-Phe cyclohexylpropionic acid 33 pCl-phe pCl-phe4-methyl-1- cyclohexylcarboxylic acid 34 pF-Phe pF-Phe 4-(tert-butyl)cyclohexylcarboxylic acid 35 pF-phe pF-phe 2-norbornylacetic acid36 Lys(Ac) Lys(Ac) 1-adamantaneacetic acid 37 Pya Pya 2-ethylbutyric 38pya pya 3,3-diphenylpropionic acid 39 Chg Chg 2-methyl-4-nitro-1-imidazolepropionic acid 40 chg chg cyclopentylacetic acid 41 tBu-GlytBu-Gly indole-3-acetic acid 42 pNH₂-Phe pNH₂-Phe (Fmoc) (Fmoc) 43pNH₂-phe pNH₂-phe (Fmoc) (Fmoc) 44 Tyr(Et) Tyr(Et) 45 tyr(Et) tyr(Et) 46pI-Phe Asp(Fm) 47 pI-phe asp(Fm) 48 Tyr(Me) pI-Phe 49 tyr(Me) pI-phe 50Tyr(Me) 51 tyr(Me) *lower case lettering indicates D-amino acids

Pools of libraries were prepared in the positional scan format. Atypical procedure for the combinatorial synthesis of the subjectbicyclic guanidine combinatorial library was as follows. One hundred mgof p-methylbenzhydrylamine (MBHA) resin (0.81 meq/g, 100-200 mesh) wascontained within a sealed polypropylene mesh packet. Followingneutralization with 5% diisopropylethylamine (DIEA) in dichloromethane(DCM), the resin was washed with DCM. The first amino acid, which wasBoc-protected, (6×) was coupled using the conventional reagentshydroxybenzotriazole (HOBt)(6×) and diisopropylcarbodiimide (DICI)(6×)(0.1 M final concentration in DMF) using the predetermined rates setforth above ([Boc-Xaa-OH] generating the R¹ group, as shown in FIG. 1).Following removal of the protecting group with 55% trifluoroacetic acid(TFA) in DCM, the packet was washed, neutralized and the second aminoacid, which also was Boc-protected, was coupled again under the sameconditions using the above predetermined rates ([Boc-Xaa-OH] generatingthe R² group, as shown in FIG. 1). Following removal of the Boc group,the dipeptide was individually acylated with a carboxylic acid in thepresence of diisopropylcarbodiimide (DICI) and 1-hydroxybenzotriazole(HOBt) utilizing the above predetermined residues once again.

The reductions were performed in 50 ml kimax tubes under nitrogen. Boricacid (40×) and trimethyl borate (40×) were added, followed by 1M BH₃-THF(40×) The tubes were heated at 65° C. for 72 h, followed by quenchingwith MeOH. The resin was then washed with tetrahydrofuran and methanol.The amine-borane complex was disassociated by overnight treatment withpiperidine at 65° C.

The cyclization occurred following treatment of the reduced acylateddipeptide with thiocarbonyldiimidazole (0.5 M in anhydrousdichloromethane) for 15 minutes followed by decantation of the solution,addition of anhydrous DCM, followed by shaking for 16 hours. Thiscyclization procedure was then repeated to ensure completion. Followingcleavage from the resin with anhydrous HF by the procedures of Houghtenet al. Int. J. Pep. Prot. Res., 27:673 (1986), which is incorporatedherein by reference, in the presence of anisole, the desired productswere extracted and lyophilized.

EXAMPLE II

This example provides the synthesis of a combinatorial library of thepresent invention according to Reaction Scheme II, which is shown inFIG. 2. The R¹, R², R³ and R⁴ groups varied as described above andbelow. Again, thirty-four first amino acids were used, generating atleast thirty-four R¹ groups, depending on the modifications to the sidechains. The amino acids used to generate the R¹ groups are again listedin Table 5. Thirty-four second amino acids and seventeen third aminoacids were used to generate the various respective R² and R³ groups,which amino acids are again listed in Table 5. Finally, the seventy-onecarboxylic acids used to acylate the tripeptides and generate thevarious R⁴ groups are also listed again in Table 5. Therefore, Table 5provides a summary of all the amino acids (R¹, R² and R³) and carboxylicacids (R⁴) used in the preparation of the combinatorial library.

TABLE 5 SUMMARY OF REAGENTS USED TO GENERATE R GROUPS IN PREPAREDLIBRARIES R1 R2 R3 R4  1 Ala Ala Ala 1-phenyl-1- cyclopropyl carboxylicacid  2 Phe Phe Phe 2-phenylbutyric acid  3 Ile Ile Gly 3-phenylbutyricacid  4 Leu Leu Leu m-toluylacetic acid  5 Val Val ala* 3-fluorophenyl-acetic acid  6 Tyr(2BrZ) Tyr(2BrZ) phe 3- bromophenylacetic acid  7 ala*ala* leu (α,α,α-trifluoro- m-toluyl) acetic acid  8 phe phe Nvap-toluylacetic acid  9 ile ile nva 3-methoxyphenyl- acetic acid 10 leuleu Nle 4- bromophenylacetic acid 11 val val nle 4-methoxyphenyl- aceticacid 12 tyr(2BrZ) tyr(2BrZ) Cha 4-ethoxyphenyl- acetic acid 13 alpha-Abualpha-Abu cha 4-isobutyl-α- methylphenyl- acetic acid 14 Aib NvaTyr(OEt) 3,4- dichlorophenyl- acetic acid 15 Nva nva tyr(OEt) 3-(3,4-dimethoxyphenyl)- propionic acid 16 nva Nle Tyr(OMe) 4-biphenylaceticacid 17 Nle nle Boc-D- α-methylcinnamic tyr(OMe) acid 18 nle Nal 2-(trifluoromethyl) cinnamic acid 19 Nal nal (3,4- dimethoxyphenyl)-acetic acid 20 nal Cha 3,4- (methylenedioxy)- phenylacetic acid 21 Chacha 2-methoxycinnamic acid 22 cha Met(O)₂ Benzoic acid 23 pF-Phe pF-Phe4-chlorocinnamic acid 24 pF-phe pF-phe m-anisic acid 25 pCl-Phe pCl-Phe4- isopropylbenzoic acid 26 pCl-phe pCl-phe 4-vinylbenzoic acid 27 ChgChg 4-fluorobenzoic acid 28 chg chg 4-bromobenzoic acid 29 Tyr(OEt)Tyr(OEt) 3,4- dimethoxycinnamic acid 30 tyr(OEt) tyr(OEt) t-cinnamicacid 31 pI-Phe pI-Phe 3,4- dimethylbenzoic acid 32 pI-phe pI-phe3-fluoro-4- methylbenzoic acid 33 Tyr(OMe) Tyr(OMe) 3-bromo-4-methylbenzoic acid 34 tyr(OMe) tyr(OMe) 3-iodo-4- methylbenzoic acid 353,4- dichlorobenzoic acid 36 4-biphenyl- carboxylic acid 37 3,4-difluorobenzoic acid 38 m-toluic acid 39 phenylacetic acid 40hydrocinnamic acid 41 3-methoxy-4- methylbenzoic acid 42 4-phenylbutyricacid 43 3,4- dimethoxybenzoic acid 44 4-ethyl-4- biphenyl- carboxylicacid 45 3,4,5- trimethoxybenzoic acid 46 butyric acid 47 heptanoic acid48 isobutyric acid 49 (+/−)-2- methylbutyric acid 50 isovaleric acid 513-methylvaleric acid 52 4-methylvaleric acid 53 p-toluic acid 54p-anisic acid 55 cyclohexyl- carboxylic acid 56 cyclohexylacetic acid 57cyclohexyl- butyric acid 58 cycloheptane- carboxylic acid 59 acetic acid60 2-methyl- cyclopropane- carboxylic acid 61 cyclobutane- carboxylicacid 62 cyclopentane- carboxylic acid 63 3-cyclopentyl- propionic acid64 2-furoic acid 65 cyclohexyl- propionic acid 66 4-methyl-1-cyclohexyl- carboxylic acid 67 4-t- butylcyclohexyl- carboxylic acid 684- methylcyclohexyl acetic acid 69 tiglic acid 70 2-norbornylacetic acid71 2-thiophene- carboxylic acid *lower case lettering indicates D-aminoacids

Pools of libraries were prepared in the positional scan format. Atypical procedure for the combinatorial synthesis of the subjectbicyclic guanidine combinatorial library was as follows.p-Methylbenzhydrylamine (MBHA) resin (100 mg, 0.81 meq/g, 100-200 mesh)was contained within a sealed polypropylene mesh packet. Followingneutralization with 5% dissopropylethylamine (DIEA) in dichloromethane(DCM), the resin was washed with DCM. The first amino acid, which wasBoc-protected, (6×) was coupled using the conventional reagentshydroxybenzotriazole (HOBt)(6×) and diisopropylcarbodiimide (DICI) (6×,0.1 M final concentration in DMF) using the predetermined ratios setforth above, as necessary (generating the R¹ group, as shown in FIG. 2).Following removal of the Boc-protecting group with 55% trifluoroaceticacid (TFA) in DCM, the packet was washed, neutralized and the secondamino acid, which also was Boc-protected, was coupled again under thesame conditions using the above predetermined ratios, as necessary(generating the R² group, as shown in FIG. 2). Following removal of theBoc-protecting group of the second amino acid with 55% trifluoroaceticacid (TFA) in DCM, the packet was again washed, neutralized and thethird amino acid, which also was Boc-protected, was coupled again underthe same conditions using the above predetermined ratios, as necessary(generating the R³ group, as shown in FIG. 2).

Following removal of the third Boc group, the tripeptide was reducedwith borane (BH₃) in THF for four days. Specifically, the reductionswere performed under nitrogen. Boric acid (40×) and trimethyl borate(40×) were added, followed by 1M BH₃-THF (40×). The resin was heated at65° C. for 72 h, followed by quenching with MeOH. The resin was thenwashed with tetrahydrofuran and methanol. On the fourth day, theamine-borane complex was disassociated by overnight treatment withpiperidine at 65° C.

Following the reduction, the N-terminus of the tripeptide was protectedby a triphenylmethyl group by overnight treatment with a solution of0.1M trityl chloride (5×) in DCM/DMF (9:1) in the presence of DIEA(25×). The three remaining secondary amines were then cyclized to form abicyclic guanidine by treatment with 0.5M 1,1-thiocarbonyldiimidazole(CSIm₂) in anhydrous dichloromethane (DCM) twice for 16 hours.

The resin was then washed with DCM three times and the trityl chlorideremoved with 2% TFA. The free N-terminus of the guanidine was thenacylated with a carboxylic acid in the presence ofdiisopropylcarbodiimide (DICI) and 1-hydroxybenzotriazole (HOBt)utilizing the above predetermined residues once again. To rid theguanidine of any HOBt salts and to encourage complete cyclization, theresin was treated with 20% piperidine in DMF for one hour and then fourmore times for five minutes each. Following final washings, the acylatedbicyclic guanidine was treated with anhydrous HF for six hours to cleaveit from the resin by the procedures of Houghten et al., Int. J. Pep.Prot. Res., 27:673 (1986), in the presence of anisole. The desiredproduct was then extracted with 95% AcOH and lyophilized.

EXAMPLE III

Following the procedures of Example I, the following pools of librariescontaining the bicyclic guanidines were prepared by the positional scanformat according to the reaction scheme shown in FIG. 1. Therefore, theR groups and their respective pool reference numbers are identified inTable 6 below. Each of the 141 pools were screened in an anti-microbialassay, σ and κ-opioid receptor assays, as provided in Example IV, and ina CaMPDE assay, as provided in Examples IX to XI. In addition, poolswere screened in an antifungal assay as provided in Example VII. ThisExample and Table 6 are provided for further reference for poolcompositions in relation to the biological data in ensuing Examples IV,VII and IX to XI.

TABLE 6 LIBRARY POOL REFERENCE NUMBERS AND REAGENTS USED TO GENERATEVARIABLE R GROUPS FOR BICYCLIC GUANIDINE LIBRARY Pool No. R¹ R² R³  1 XX 3-phenylbutyric acid  2 X X m-toluylacetic acid  3 X X3-fluorophenylacetic acid  4 X X p-toluylacetic acid  5 X X4-fluorophenylacetic acid  6 X X 3-methoxyphenylacetic acid  7 X X4-methoxyphenylacetic acid  8 X X 4-ethoxyphenylacetic acid  9 X X3-(3,4-dimethoxyphenyl)- propionic acid 10 X X 4-biphenylacetic acid 11X X (3,4-dimethoxyphenyl)acetic acid 12 X X phenylacetic acid 13 X Xhydrocinnamic acid 14 X X 4-phenylbutyric acid 15 X X butyric acid 16 XX heptanoic acid 17 X X isobutyric acid 18 X X (+/−)-2-methylbutyricacid 19 X X isovaleric acid 20 X X 3-methylvaleric acid 21 X X4-methylvaleric acid 22 X X (tert-butyl)acetic acid 23 X Xcyclohexylcarboxylic acid 24 X X cyclohexylacetic acid 25 X Xcyclohexylbutyric acid 26 X X cycloheptanecarboxylic acid 27 X X lacticacid 28 X X acetic acid 29 X X cyclobutanecarboxylic acid 30 X Xcyclopentanecarboxylic acid 31 X X 3-cyclopentylpropionic acid 32 X Xcyclohexylpropionic acid 33 X X 4-methyl-1- cyclohexylcarboxylic acid 34X X 4-(tert- butyl)cyclohexylcarboxylic acid 35 X X 2-norbornylaceticacid 36 X X 1-adamantaneacetic acid 37 X X 2-ethylbutyric 38 X X3,3-diphenylpropionic acid 39 X X 2-methyl-4-nitro-1- imidazolepropionicacid 40 X X cyclopentylacetic acid 41 X X indole-3-acetic acid 42 X AlaX 43 X Phe X 44 X Gly X 45 X Ile X 46 X Lys(Clz) X 47 X Leu X 48 XMet(O) X 49 X Arg(Tos) X 50 X Val X 51 X Tyr(Brz) X 52 X ala* X 53 X pheX 54 X ile X 55 X lys(Clz) X 56 X leu X 57 X arg(Tos) X 58 X val X 59 Xtyr(Brz) X 60 X α-Abu X 61 X Nve X 62 X nve X 63 X Nle X 64 X nle X 65 XOrn(Cbz) X 66 X Nap X 67 X nap X 68 X Cha X 69 X cha X 70 X Met(O₂) X 71X pNO₂-Phe X 72 X pNO₂-phe X 73 X pCl-Phe X 74 X pCl-phe X 75 X pF-Phe X76 X pF-phe X 77 X Lys(Ac) X 78 X Pya X 79 X pya X 80 X Chg X 81 X chg X82 X tBu-Gly X 83 X pNH₂-Phe X (Fmoc) 84 X pNH₂-phe X (Fmoc) 85 XTyr(Et) X 86 X tyr(Et) X 87 X Asp(Fm) X 88 X asp(Fm) X 89 X pI-Phe X 90X pI-phe X 91 X Tyr(Me) X 92 X tyr(Me) X 93 Ala X X 94 Phe X X 95 Ile XX 96 Lys(Clz) X X 97 Leu X X 98 Met(O) X X 99 Arg(Tos) X X 100  Val X X101  Tyr(Brz) X X 102  ala* X X 103  phe X X 104  ile X X 105  lys(Clz)X X 106  leu X X 107  arg(Tos) X X 108  val X X 109  tyr(Brz) X X 110 α-Abu X X 111  α-Aib X X 112  Nve X X 113  nve X X 114  Nle X X 115  nleX X 116  Orn(Cbz) X X 117  Nap X X 118  nap X X 119  Cha X X 120  cha XX 121  Met(O₂) X X 122  pNO₂-Phe X X 123  pNO₂-phe X X 124  pCl-Phe X X125  pCl-phe X X 126  pF-Phe X X 127  pF-phe X X 128  Lys(Ac) X X 129 Pya X X 130  pya X X 131  Chg X X 132  chg X X 133  tBu-Gly X X 134 pNH₂-Phe X X (Fmoc) 135  pNH2-phe X X (Fmoc) 136  Tyr(Et) X X 137 tyr(Et) X X 138  pI-Phe X X 139  pI-phe X X 140  Tyr(Me) X X 141 tyr(Me) X X *lower case lettering indicates D-amino acids

EXAMPLE IV

This example describes initial biological screens of all 141combinatorial library pools as identified in the above Example III. Morespecifically, this example provides an initial screen of all thebicyclic guanidines in (1) the σ receptor assay and (3) κ-opioidreceptor assay, each of which are described in detail above. The resultsof those screens are provided in Table 7 below. In addition, the resultsof the σ-receptor and κ-opioid receptor assays are depicted graphicallyin FIGS. 2 and 3.

The results of these assays evidence that many of the bicyclic guanidinecompounds contained within the libraries are biologically active, asinhibitor of a specific receptors. Moreover, the results of the screensprovide evidence that there is selectivity of certain compounds for onereceptor over another.

TABLE 7 Radio-Receptor Assays Of The Bicyclic Guanidine Library(Positional Scanning Format) κ-Opioid Receptor Pool σ Receptor AssayAssay No. (% Bound) (% Bound)  1 51.15 38.63  2 53.21 36.77  3 64.0089.29  4 70.19 22.10  5 61.49 33.13  6 72.31 24.68  7 60.94 24.52  866.83 33.37  9 103.06 42.31 10 82.59 46.42 11 115.47 65.74 12 68.9039.16 13 42.31 25.43 14 27.38 30.55 15 55.39 37.80 16 33.19 30.12 1769.62 19.29 18 58.76 23.29 19 36.43 33.03 20 20.40 27.43 21 25.17 25.3822 19.00 17.06 23 26.44 21.98 24 13.46 10.35 25 8.77 8.38 26 21.79 11.8727 97.95 49.99 28 50.31 29.67 29 58.83 17.50 30 38.62 16.12 31 16.577.10 32 10.57 4.68 33 26.00 18.96 34 17.63 22.53 35 11.98 13.36 36 15.357.56 37 41.34 15.87 38 90.79 41.08 39 99.47 37.47 40 17.82 27.02 41108.67 39.85 42 8.95 17.02 43 59.03 26.56 44 5.78 28.79 45 65.43 15.4146 88.96 46.32 47 67.95 27.56 48 80.10 41.32 49 36.84 41.08 50 10.4726.58 51 64.10 29.36 52 6.91 40.91 53 77.93 31.14 54 95.74 22.03 5571.63 44.87 56 93.75 14.61 57 116.01 44.73 58 23.86 20.25 59 96.76 20.2560 17.11 21.21 61 29.32 23.06 62 33.70 14.86 63 69.11 27.90 64 61.2815.98 65 115.70 47.10 66 84.81 31.32 67 78.50 23.64 68 86.83 36.61 6988.92 34.13 70 116.29 72.27 71 104.24 67.83 72 92.44 47.61 73 66.9437.28 74 66.45 24.39 75 71.45 34.77 76 73.67 35.31 77 99.72 48.10 7894.78 44.88 79 86.51 30.66 80 102.50 9.99 81 77.20 21.18 82 86.61 17.2583 110.16 33.32 84 102.04 34.70 85 66.37 41.40 86 73.28 20.55 87 104.5476.93 88 89.65 53.16 89 73.05 45.38 90 58.13 26.33 91 42.42 9.81 9252.95 13.60 93 26.12 2.03 94 11.98 22.25 95 15.29 15.87 96 47.37 37.7897 9.41 12.18 98 23.82 28.10 99 43.85 54.16 100  13.53 17.10 101  27.4918.58 102  24.85 33.32 103  17.22 22.96 104  16.44 13.52 105  42.2948.41 106  9.91 19.25 107  38.54 66.20 108  19.28 16.46 109  28.96 24.20110  22.18 4.65 111  28.75 26.69 112  12.66 12.28 113  17.32 24.68 114 6.84 14.29 115  7.26 18.39 116  56.84 43.12 117  25.88 47.86 118  26.7344.37 119  6.41 18.78 120  6.70 5.38 121  64.65 89.42 122  36.72 63.14123  35.57 64.93 124  27.67 56.90 125  36.13 57.95 126  34.41 32.00 127 25.09 39.06 128  93.55 40.86 129  89.78 56.15 130  82.81 38.00 131 12.55 5.61 132  24.16 0.51 133  84.38 50.78 134  120.65 53.21 135 118.48 38.12 136  62.93 38.52 137  57.04 37.86 138  57.38 67.66 139 49.87 59.78 140  44.99 39.33 141  40.48 26.56

EXAMPLE V

From the above initial biological screens of all 141 combinatoriallibrary pools, twenty-four individual compounds were synthesizedseparately and retested in the same assays to determine IC₅₀ valuesusing similar conditions to those of Example I. The various R¹, R², andR³ groups used in these individual synthesis are set forth below inTable 8. In summary, the twenty four individual compounds synthesizedand tested were those compounds derived from every permutation of thefollowing amino acids and carboxylic acids.

TABLE 8 SUMMARY OF R GROUPS FOR TWENTY-FOUR INDIVIDUAL COMPOUND S R¹ R²R³ Ala O-Me-Tyr cyclohexylpropionic acid chg O-Me-tyr*1-adamantaneacetic acid Chg leu chg *lower case lettering indicatesD-amino acids

A typical procedure for the synthesis of an individual compound is asfollows. One hundred mg of p-methylbenzhydrylamine (MBHA) resin (0.81meq/g, 100-200 mesh) was contained within a sealed polypropylene meshpacket. Following neutralization with 5% diisopropylethylamine (DIEA) indichloromethane (DCM), the resin was washed with DCM. The first aminoacid coupled using the conventional reagents hydroxybenzotriazole(HOBt)(6×) and diisopropylcarbodiimide (DICI)(6×) (0.1 M finalconcentration in DMF). Following removal of the protecting group with55% trifluoroacetic acid (TFA) in DCM, the packet was washed,neutralized and the second amino acid coupled under the same conditionsas for the first amino acid. Following removal of the Boc group, thedipeptide was individually acylated with a carboxylic acid in thepresence of diisopropylcarbodiimide (DICI) and 1-hydroxybenzotriazole(HOBt) under the same conditions as for the first amino acid.

Reduction was performed in a 50 ml kimax tube under nitrogen. Boric acid(40×) and trimethyl borate (40×) were added, followed by 1MBH₃-THF(40×). The tubes were heated at 65° C. for 72 h, followed byquenching with MeOH. The resin was then washed with tetrahydrofuran andmethanol. The amine-borane complex was disassociated by overnighttreatment with piperidine at 65° C.

Cyclization occurred following treatment of the reduced acylateddipeptide with thiocarbonyldiimidazole (0.5 M in dichloromethaneanhydrous) for 15 minutes followed by decantation of the solution,addition of anhydrous DCM, followed by shaking for 16 hours. Thiscyclization procedure was repeated to ensure completion. Followingcleavage from the resin with anhydrous HF by the procedures of Houghtenet al. Int. J. Pep. Prot. Res., 27:673 (1986), which is incorporatedherein by reference, in the presence of anisole, the desired productswere extracted and lyophilized. The desired product was obtained in goodyield and purity following lyophilization.

The twenty-four individual bicyclic guanidine compounds were testedwith: (1) the σ receptor assay; and (2) κ-opioid receptor assay, asdescribed above. As provided in Tables 9 and 10, the results of theseassays evidence that the individual compounds are inhibitors of theκ-opioid and σ receptors and have significant biologically activity. Thecompounds are provided below from most active to least active.

TABLE 9 K-OPIOID RECEPTOR ASSAY FOR 24 INDIVIDUAL COMPOUNDS Cmpd. IC₅₀No. R¹ R² R³ (nM)  1 Ala Tyr(Me) cyclohexylpropionic acid  37 16 AlaTyr(Me) adamantaneacetic acid  238 17 chg Tyr(Me) adamantaneacetic acid 341  4 Ala Tyr(Me) cyclohexylpropionic acid  502  5 chg Tyr(Me)cyclohexylpropionic acid  547 21 Chg leu adamantaneacetic acid 1206 24Chg Chg adamantaneacetic acid 1492 13 Ala Tyr(Me) adamantaneacetic acid1523 20 chg leu adamantaneacetic acid 1747  8 chg leucyclohexylpropionic acid 1767 22 Ala Chg adamantaneacetic acid 1941 10Ala Chg cyclohexylpropionic acid 2479  2 chg Tyr(Me) cyclohexylpropionicacid 3456 19 Ala leu adamantaneacetic acid 3641  9 Chg leucyclohexylpropionic acid 3744  3 Chg Tyr(Me) cyclohexylpropionic acid3872 12 Chg Chg cyclohexylpropionic acid 4482 15 Chg Tyr(Me)adamantaneacetic acid 4923  6 Chg Tyr(Me) cyclohexylpropionic acid 5026 7 Ala leu cyclohexylpropionic acid 5436 11 chg Chg cyclohexylpropionicacid 10333  18 Chg Tyr(Me) adamantaneacetic acid >10333  14 chg Tyr(Me)adamantaneacetic acid >10333  23 chg Chg adamantaneacetic acid >10333 

TABLE 10 σ-RECEPTOR ASSAY FOR 24 INDIVIDUAL COMPOUNDS Cmpd. IC₅₀ No. R¹R² R³ (nM)  9 Chg leu cyclohexylpropionic acid  13  3 Chg Tyr(Me)cyclohexylpropionic acid  23  5 chg Tyr(Me) cyclohexylpropionic acid  42 4 Ala Tyr(Me) cyclohexylpropionic acid  52 17 chg Tyr(Me)adamantaneacetic acid  56  6 Chg Tyr(Me) cyclohexylpropionic acid  68  1Ala Tyr(Me) cyclohexylpropionic acid  94 21 Chg leu adamantaneaceticacid 124  8 chg leu cyclohexylpropionic acid 201 12 Chg Chgcyclohexylpropionic acid 210 13 Ala Tyr(Me) adamantaneacetic acid 235 15Chg Tyr(Me) adamantaneacetic acid 256 20 chg Leu adamantaneacetic acid267 18 Chg Tyr(Me) adamantaneacetic acid 297 10 Ala Chgcyclohexylpropionic acid 348 11 chg Chg cyclohexylpropionic acid 405  7Ala leu cyclohexylpropionic acid 530  2 chg Tyr(Me) cyclohexylpropionicacid 585 14 chg Tyr(Me) adamantaneacetic acid 823 24 Chg Chgadamantaneacetic acid 930 19 Ala leu adamantaneacetic acid 974 22 AlaChg adamantaneacetic acid 1025  16 Ala Tyr(Me) adamantaneacetic acid1077  23 chg Chg adamantaneacetic acid 1577 

EXAMPLE VI

An additional seventeen individual compounds were synthesized and testedfor activity in the σ-receptor assay. The individual compounds weresynthesized following the same procedures as provided above in Example Vand using the amino acids and carboxylic acids listed in Table 11 below.The IC₅₀ values were determined as detailed above. The results providedin Table 11 below evidence the significant biological activity of thecompounds.

TABLE 11 σ-RECEPTOR ASSAY FOR 17 INDIVIDUAL COMPOUNDS R¹ R² R³ IC₅₀(nM)Phe Nal acetic acid  22 Phe Tyr(Et) acetic acid  33 Phe nal acetic acid 42 Phe Cha acetic acid  52 Phe pCl-Phe acetic acid  60 Phe Tyr(Et)acetic acid  64 Phe pI-Phe acetic acid  65 Phe Tyr(Me) acetic acid  69Phe pNO₂-Phe acetic acid 102 Phe Phe (tert- 112 butyl)acetic acid Phechg acetic acid 119 Phe pCl-phe acetic acid 127 Phe Phe isovaleric 156acid Phe Lys(Ac) acetic acid 202 Nle Phe acetic acid 214 Phe pF-Pheacetic acid 215 Phe pF-phe acetic acid 242

EXAMPLE VII

This example describes initial antifungal screens of combinatoriallibrary pools identified in Example III. In addition, the results of theantifungal assay is depicted in FIG. 5. The results of this assayevidences that many of the bicyclic guanidine compounds contained withinthe libraries are biologically active as antifungal agents.

TABLE 12 Anti-Fungal Assay of a Bicyclic Guanidine Library Pool No.IC₅₀(μg/ml) MIC(μg/ml) 119  18 20-32 120  19 25-32 132  34 40-62 131  3745-62 126  94 >250 127  97 125-250 115 105 >250 128 145 >250 112161 >250  95 170 >250  96 172 >250 113 176 >250  97 176 >250 111180 >250 106 181 200-250 103 182 >250 124 182 >250 104 185 200-250 141188 >250 108 194 >250 125 195 >250  98 197  250 136 199 >250 116199 >250 100 200 >250 105 206 >250  94 229 >250 134 236 >250 135240 >250 other >250  >250 pools  68  20 32-62  69  23 32-62  80  48 70-125  81  78 125-250  54  80 125-250  74  88 >250  44 138 150-250  56169 >250  47 172 >250  85 175 >250  64 177 >250  86 181 >250  63182 >250  73 182 >250  58 185 >250  61 187 >250  43 189 >250  82191 >250  83 193 >250  62 201 >250  91 201 >250  84 208 >250  45210 >250  53 211 >250  75 211 >250  74 215 >250  72 224 >250  89225 >250 other >250  >250 pools  34  19 32-62  36  22 32-64  25  4050-62  35  54 125-250  16  80 125-250  31  80 125-250  31  80 125-250 33  82 125-250  24  85 125-250  32 100 200-250  20 119 >250  21121 >250  26 142 >250  13 144 >250  40 155 >250  22 202 >250  14203 >250  23 208 >250  37 214 >250  30 218 >250  1 220 >250  4 227 >250 3 229 >250  6 234 >250 other >250  >250 pools

EXAMPLE VIII

From the above initial screens of all 141 combinatorial library pools,thirty-two individual compounds were synthesized separately underprocedures similar to those described in Example V. These compounds wereretested in the same antifungal assay to determine IC₅₀ and MIC valuesas set forth below in Table 13. The various R groups used are also setforth in Table 13.

TABLE 13 Anti-Fungal Activity Assay for 32 Individual Compounds R¹ R² R³IC₅₀(μg/ml) MIC(μg/ml) cha Cha 4-(tert-butyl)- 2.34 3-4 cyclohexyl-carboxylic acid chg Cha 1-adamantaneacetic 2.40 3-4 acid Chg cha1-adamantaneacetic 2.52 3-4 acid cha Cha 1-adamantaneacetic 2.92 4-8acid Cha cha 1-adamantaneacetic 3.00 4-8 acid chg Cha 4-(tert-butyl)-3.53 4-8 cyclohexyl- carboxylic acid Cha Cha 4-(tert-butyl)- 4.22 5-8cyclohexyl- carboxylic acid Cha cha 4-(tert-butyl)- 4.39 5-8 cyclohexyl-carboxylic acid Cha Cha 1-adamantaneacetic 4.42 5-8 acid cha cha4-(tert-butyl)- 4.54 5-8 cyclohexyl- carboxylic acid Chg cha4-(tert-butyl)- 4.57 5-8 cyclohexyl- carboxylic acid chg cha4-(tert-butyl)- 6.61  8-16 cyclohexyl- carboxylic acid cha cha1-adamantaneacetic 7.29  8-16 acid Cha Chg 4-(tert-butyl)- 7.31  8-16cyclohexyl- carboxylic acid cha Chg 1-adamantaneacetic 8.27 10-16 acidChg Cha 4-(tert-butyl)- 8.71 10-16 cyclohexyl- carboxylic acid cha chg4-(tert-butyl)- 8.75 10-16 cyclohexyl- carboxylic acid chg Chg1-adamantaneacetic 9.01 10-16 acid cha chg 1-adamantaneacetic 9.12 10-16acid Cha chg 4-(tert-butyl)- 9.19 10-16 cyclohexyl- carboxylic acid ChaChg 1-adamantaneacetic 9.29 10-16 acid Cha chg 1-adamantaneacetic 9.3610-16 acid cha Chg 4-(tert-butyl)- 9.67 16-32 cyclohexyl- carboxylicacid Chg chg 1-adamantaneacetic 9.92 11-16 acid chg Chg 4-(tert-butyl)-16.80 18-32 cyclohexyl- carboxylic acid Chg Chg 1-adamantaneacetic 18.5820-32 acid chg chg 1-adamantaneacetic 23.13 32-64 acid chg cha1-adamantaneacetic 23.79 32-64 acid Chg Chg 4-(tert-butyl)- 24.07 32-64cyclohexyl- carboxylic acid Chg chg 4-(tert-butyl)- 29.34 >62cyclohexyl- carboxylic acid chg chg 4-(tert-butyl)- 34.28 >62cyclohexyl- carboxylic acid Chg Cha 1-adamantaneacetic 38.77 >62 acid*lower case indicates D-amino acid

EXAMPLE IX

This example describes initial screens of all 141 combinatorial librarypools identified in Example III for activity as calmodulin antagonists.The results of the screens provided in Table 14 below. In addition, theresults of the CaMPDE assay is depicted in FIG. 6. The results of thisassay evidences that many of the bicyclic guanidine compounds containedwithin the libraries are biologically active as calmodulin antagonists.

TABLE 14 CaMPDE Assay of a Bicyclic Guanidine Library (PositionalScanning Format) Pool No. % Inhibition 117 ≧100 118 ≧100 138 ≧100 126≧100 125 ≧100 107 ≧100 139 ≧100 127 99.6 122 97.2 123 94.6 119 91.9 13586.7 124 85.9  94 82.3 134 78.4 103 75.8 120 69.0 137 61.3 110 56.3 10154.7  97 54.5  95 53.3 140 48.4  98 47.0 105 46.8 114 41.6  96 39.8 10438.6 141 34.3 129 34.1 132 31.5 128 28.3 106 27.3 112 26.3 100 25.5 11522.9  93 21.5 116 21.5 108 21.0 121 18.8 110 17.4 133 8.6 102 6.7  67≧100  66 ≧100  57 ≧100  74 ≧100  69 ≧100  89 ≧100  73 ≧100  68 ≧100  75≧100  72 ≧100  90 97.8  80 96.5  71 92.1  53 90.8  49 88.9  43 88.5  8188.2  76 82.5  54 81.6  91 71.8  56 68.6  47 67.7  45 66.1  64 65.6  7065.0  82 61.8  85 55.4  62 52.4  86 47.6  55 46.0  84 45.3  83 42.8  4639.8  50 38.2  65 38.2  51 37.3  60 34.8  61 30.5  42 29.8  52 29.1  8727.3  58 26.4  48 20.0  78 15.4  79 9.2  77 7.0  44 0  88 0  32 ≧100  10≧100  34 ≧100  25 ≧100  31 ≧100  36 ≧100  26 ≧100  8 ≧100  14 ≧100  24≧100  35 ≧100  40 ≧100  23 99.9  5 99.4  6 99.4  33 96.5  39 89.4  1388.9  21 88.9  17 88.0  22 82.8  30 82.8  3 80.9  2 79.8  12 79.6  178.7  7 77.3  4 73.6  37 73.6  18 68.4  15 58.3  9 54.7  29 50.8  2837.8  19 28.7

EXAMPLE X

After the initial screen, IC₅₀ values were determined for combinatoriallibrary pools with the most activity. These results are provided inTable 15 below.

TABLE 15 CaMPDE Assay of a Bicyclic Guanidine Library (PositionalScanning Format) (Determination of IC₅₀ Values) Pool No. IC₅₀ (μg/ml)126 5.0 138 5.3 118 5.3 136 6.4  99 8.3 134 8.8 120 9.1 117 9.1 131 9.4135 9.7 103 10.6 139 10.6 123 10.9 107 11.1 137 11.3 127 11.3 119 11.9 94 12.2 111 12.5  97 12.8  95 13.3 101 14.7  66 3.9  67 4.7  89 4.7  755.8  49 7.0  43 8.4  68 8.7  69 8.8  82 9.0  81 9.2  84 9.3  80 10.2  5710.3  74 10.4  64 11.6  53 11.8  76 11.9  83 12.1  56 12.2  54 12.3  4512.8  92 14.4  85 14.6  62 16.1  70 17.6  59 17.9  47 18.0  63 26.4  323.1  10 3.4  38 4.9  34 5.0  25 5.3  13 7.2  3 7.8  36 7.9  35 8.0  68.1  37 8.6  41 9.2  26 10.1  16 10.2  5 10.2  4 10.4  8 10.5  1 10.7 23 10.9  24 10.9  33 11.2  21 11.4  40 12.0  30 12.1  22 12.2  39 12.2 7 13.3  20 13.4  18 14.3  15 14.5  11 15.8  29 16.8  27 18.5  9 19.0

EXAMPLE XI

The active mixtures listed in Table 15 above were also screened forspecificity to calmodulin versus phosphodiesterase. These results areprovided in Table 16 below.

TABLE 16 Specificity To CaM versus PDE of a Bicyclic Guanidine Libraryat 15 μg/ml (Positional Scanning Format) CaM PDE Pool No. (% inhibition)(% inhibition) Specificity 117 115.5 13.9 8.3 118 114.9 23.7 4.8 138109.7 30.9 3.5 126 107.9 27.2 4.0 125 106.4 25.4 4.2 107 101.4 28.3 3.6139 101.0 32.7 3.1 125 99.6 15.6 6.4 122 97.2 24.9 3.9 123 94.6 21.1 4.5119 91.9 24.3 3.8 135 86.7 41.6 2.1 124 85.9 24.3 3.5  94 82.3 15.6 5.3134 78.4 31.5 2.5 103 75.8 15.0 5.0  67 129.8 28.3 4.6  66 123.9 31.83.9  89 115.9 23.7 4.9  73 115.0 35.0 3.3  68 107.4 27.2 4.0  75 107.448.8 2.2  72 107.0 28.0 3.8  90 97.8 33.2 2.9  80 96.5 22.8 4.2  71 92.130.1 3.1  53 90.8 17.3 5.2  49 88.9 31.5 2.8  43 88.5 15.0 5.9  81 88.217.6 5.0  76 82.5 20.5 4.0  54 81.6 9.5 8.6  32 149.2 64.5 2.3  10 140.556.9 2.5  34 137.8 32.4 4.3  25 134.4 36.1 3.7  38 132.5 17.9 7.4  41128.7 33.2 3.9  16 119.1 48.6 2.5  31 119.1 30.9 3.9  36 116.3 45.1 2.6 26 115.9 34.7 3.3  8 114.5 36.1 3.2  40 100.6 17.6 5.7  23 99.9 35.02.9  5 99.4 9.8 10.1  6 99.4 35.3 2.8  33 96.5 28.3 3.4  39 89.4 35.02.6  13 88.9 52.6 1.7  21 88.9 20.5 4.3  17 88.0 32.9 2.7  22 82.8 22.53.7  30 82.8 29.2 2.8  3 80.9 26.6 3.0  2 79.8 33.8 2.4  12 79.6 43.91.8  1 78.7 37.3 2.1  7 77.3 29.5 2.6  4 73.6 25.4 2.9  37 73.6 19.1 3.9

EXAMPLE XII

From the above screens, fifty-three individual compounds were assayedand their IC₅₀ values determined. These results, as well as eachcompounds R groups, are provided in Table 17 below.

TABLE 17 CaMPDE Assay for 53 Individual Compounds IC₅₀ R¹ R² R³ (μg/ml)L-Cha D-cha 4-(tert-butyl)- 0.86 cyclohexylcarboxylic acid L-Cha L-Cha4-(tert-butyl)- 1.02 cyclohexylcarboxylic acid L-Cha D-cha1-adamantaneacetic acid 1.05 L-Chg D-cha 4-(tert-butyl)- 1.06cyclohexylcarboxylic acid D-cha L-Cha 4-(tert-butyl)- 1.28cyclohexylcarboxylic acid L-Chg D-cha 1-adamantaneacetic acid 1.28 D-chgL-Cha 4-(tert-butyl)- 1.41 cyclohexylcarboxylic acid L-Cha D-chg4-(tert-butyl)- 1.76 cyclohexylcarboxylic acid D-cha L-Cha1-adamantaneacetic acid 1.85 L-Chg D-chg 1-adamantaneacetic acid 2.02D-cha D-cha 4-(tert-butyl)- 2.02 cyclohexylcarboxylic acid D-cha L-Chg4-(tert-butyl)- 2.08 cyclohexylcarboxylic acid D-cha D-chg4-(tert-butyl)- 2.09 cyclohexylcarboxylic acid D-chg L-Cha1-adamantaneacetic acid 2.14 L-Chg L-Cha 4-(tert-butyl)- 2.30cyclohexylcarboxylic acid L-Cha D-chg 1-adamantaneacetic acid 2.76 D-chgL-Cha 1-adamantaneacetic acid 2.92 L-Phe L-Phe cyclohexylbutyric acid3.00 L-Cha L-Cha 1-adamantaneacetic acid 3.66 D-chg D-cha4-(tert-butyl)- 3.82 cyclohexylcarboxylic acid L-Cha L-Chg4-(tert-butyl)- 4.45 cyclohexylcarboxylic acid D-cha L-Chg1-adamantaneacetic acid 4.57 D-chg L-Chg 4-(tert-butyl)- 4.58cyclohexylcarboxylic acid D-cha D-cha 1-adamantaneacetic acid 4.75 L-PheL- acetic acid 5.48 Asp(Fm) L-Chg D-chg 4-(tert-butyl)- 5.61cyclohexylcarboxylic acid D-chg L-Chg 1-adamantaneacetic acid 5.66 L-ChgO- cyclohexylpropionic acid 5.77 tyr(Me) L-Phe L-Phe2,4-dinitrophenylacetic 6.06 acid L-Chg L-Cha 1-adamantaneacetic acid6.07 L-Chg O- 1-adamantaneacetic acid 6.21 tyr(Me) L-Chg L-Chg4-(tert-butyl)- 6.25 cyclohexylcarboxylic acid D-chg O-cyclohexylpropionic acid 6.28 tyr(Me) D-chg L-Cha cyclohexylpropionicacid 6.48 D-chg D-chg 4-(tert-butyl)- 6.61 cyclohexylcarboxylic acidL-Phe L-Phe 4-biphenylacetic acid 6.65 D-chg D-cha 1-adamantaneaceticacid 6.80 D-chg O- cyclohexylpropionic acid 6.92 Tyr(Me) L-Chg D-leu1-adamantaneacetic acid 6.96 L-Phe D-trp acetic acid 7.00 L-Cha L-Chg1-adamantaneacetic acid 7.32 L-Phe L-Phe 1-adamantaneacetic acid 7.56D-cha D-chg 1-adamantaneacetic acid 7.85 L-Chg L-Chg 1-adamantaneaceticacid 8.65 L-Phe L-Trp acetic acid 8.88 L-Phe L-Phe cyclohexylpropionicacid 8.93 D-chg D-chg 1-adamantaneacetic acid 9.39 L-Chg D-leucyclohexylpropionic acid 9.41 L-Phe L-Phe 4-(tert-butyl)- 9.53cyclohexylcarboxylic acid D-ala L-Phe acetic acid 9.75 L- L-Phe aceticacid 9.79 Met(O) L- L-Phe acetic acid 10.70 Formyl- Trp L-Chg L-Cha1-adamantaneacetic acid 11.66

All journal article and reference citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the inventions. Accordinglythe invention is limited only by the claims.

We claim:
 1. A single bicyclic guanidine compound of the structure:

wherein: R¹ is selected from the groups consisting of C₁ to C₁₀ alkyl,C₁ to C₁₀ substituted alkyl, C₇ to C₁₆ phenylalkyl, C₇ to C₁₆substituted phenylalkyl, phenyl, substituted phenyl, C₃ to C₇cycloalkyl, C₃ to C₇ substituted cycloalkyl, benzyl, and substitutedbenzyl; R² is selected from the groups consisting of C₁ to C₁₀ alkyl, C₁to C₁₀ substituted alkyl, C₇ to C₁₆ phenylalkyl, C₇ to C₁₆ substitutedphenylalkyl, phenyl, substituted phenyl, C₃ to C₇ cycloalkyl, C₃ to C₇substituted cycloalkyl, benzyl, substituted benzyl, naphthyl, andsubstituted naphthyl; and R³ is selected from the group consisting of ahydrogen atom, C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₁ to C₁₀ substitutedalkyl, C₂ to C₁₀ alkynyl, C₃ to C₇ substituted cycloalkyl, C₃ to C₇cycloalkenyl, C₃ to C₇ substituted cycloalkenyl, C₇ to C₁₆ phenylalkyl,C₇ to C₁₆ substituted phenylalkyl, C₇ to C₁₆ phenylalkenyl and C₇ to C₁₆substituted phenylalkenyl; or a pharmaceutically-acceptable saltthereof.
 2. A single bicyclic guanidine compound of the structure:

wherein: R¹ is selected from the group consisting of methyl, benzyl,2-butyl, N-methyl,N-thiocarbonylimidazole-aminobutyl, 2-methylpropyl,methylsulfinylethyl, guanidinopropyl, 2-propyl, 4-hydroxybenzyl, ethyl,dimethyl, propyl, butyl, N-methyl,N-thiocarbonylimidazole-aminopropyl,2-naphthylmethyl, cyclohexylmethyl, methylsulfonylethyl, 4-nitrobenzyl,4-chlorobenzyl, 4-fluorobenzyl,N-ethyl,N-thiocarbonylimidazole-aminobutyl, 3-pyridylmethyl, cyclohexyl,tert-butyl, N-methyl,N-thiocarbonylimidazole-4-aminobenzyl,4-ethoxybenzyl, 4-iodobenzyl, 4-methoxybenzyl andN-(methyl)indol-3-ylmethyl; R² is selected from the group consisting ofmethyl, benzyl, hydrogen, 2-butyl,N-methyl,N-thiocarbonylimidazole-aminobutyl, 2-methylpropyl,methylsulfinylethyl, guanidinopropyl, 2-propyl, 4-hydroxybenzyl, ethyl,propyl, butyl, N-methyl,N-thiocarbonylimidazole-aminopropyl,2-naphthylmethyl, cyclohexylmethyl, methylsulfonylethyl, 4-nitrobenzyl,4-chlorobenzyl, 4-fluorobenzyl,N-ethyl,N-thiocarbonylimidazole-aminobutyl, 3-pyridylmethyl, cyclohexyl,tert-butyl, N-methyl,N-thiocarbonylimidazole-4-aminobenzyl,4-ethoxybenzyl, hydroxyethyl, 4-iodobenzyl, 4-methoxybenzyl andindol-3-ylmethyl; and R³ is selected from the group consisting of3-phenylbutyl, m-toluylethyl, 3-fluorophenylethyl, p-toluylethyl,4-fluorophenylethyl, 3-methoxyphenylethyl, 4-methoxyphenylethyl,4-ethoxyphenylethyl, 3-(3,4-dimethoxyphenyl)propyl, 4-biphenylethyl,3,4-dimethoxyphenylethyl, phenylethyl, 3-phenylpropyl, 4-phenylbutyl,butyl, heptyl, isobutyryl, (+/−)-2-methylbutyl, isovaleryl,3-methylvaleryl, 4-methylvaleryl, (tert-butyl)ethyl, cyclohexylmethyl,cyclohexylethyl, cyclohexylbutyl, cycloheptylmethyl, 2-hydroxypropyl,ethyl, cyclobutylmethyl, cyclopentylmethyl, 3-cyclopentylpropyl,cyclohexylpropyl, 4-methyl-1-cyclohexylmethyl,4-(tert-butyl)-1-cyclohexylmethyl, 2-norbornylethyl, 1-adamantylethyl,2-ethylbutyl, 3,3-diphenylpropyl, 2-methyl-4-nitro-1-imidazolylpropyl,cyclopentylethyl, 3-indolylethyl and 2,4-dinitrophenylethyl.
 3. Thecompound of claim 1, wherein: R¹ is selected from the group consistingof methyl and cyclohexyl; R² is selected from the group consisting of4-methoxybenzyl, 2-methylpropyl and cyclohexyl; and R³ is selected fromthe group consisting of 3-cyclohexylpropyl and 1-adamantylethyl.
 4. Thecompound of claim 1, wherein: R¹ is selected from the group consistingof benzyl and butyl; R² is selected from the group consisting of2-naphthylmethyl, 4-ethoxybenzyl, cyclohexylmethyl, 4-chlorobenzyl,4-iodobenzyl, 4-methoxybenzyl, 4-nitrobenzyl, benzyl, cyclohexyl,N-ethyl,N-thiocarbonylimidazole-aminobutyl, and 4-fluorobenzyl; and R³is selected from the group consisting of methyl, (tert-butyl)ethyl andisovaleryl.
 5. The compound of claim 1, wherein: R¹ is selected from thegroup consisting of cyclohexyl and cyclohexylmethyl; R² is selected fromthe group consisting of cyclohexyl and cyclohexylmethyl; and R³ isselected from the group consisting of 4-tert-butyl-1-cyclohexylmethyland 1-adamantylethyl.
 6. The compound of claim 1, wherein: R¹ isselected from the group consisting of cyclohexyl, cyclohexylmethyl,methyl, benzyl, methylsulfinylethyl and N-(methyl)indol-3-ylmethyl; R²is selected from the group consisting of cyclohexyl, cyclohexylmethyl,benzyl, hydroxyethyl, 4-methoxybenzyl, 2-methylpropyl andindol-3-ylmethyl; and R³ is selected from the group consisting of4-tert-butyl-1-cyclohexylmethyl, 1-adamantylethyl, cyclohexylbutyl,ethyl, 4-biphenylethyl and 2,4-dinitrobenzyl.
 7. A single bicyclicguanidine compound of the structure:

wherein: R¹ is selected from the group consisting of a hydrogen atom, C₁to C₁₀ alkyl, C₁ to C₁₀ substituted alkyl, C₇ to C₁₆ phenylalkyl, C₇ toC₁₆ substituted phenylalkyl, phenyl, substituted phenyl, C₃ to C₇cycloalkyl, C₃ to C₇ substituted cycloalkyl, benzyl, and substitutedbenzyl; R² is selected from the group consisting of a hydrogen atom, C₁to C₁₀ alkyl, C₁ to C₁₀ substituted alkyl, C₇ to C₁₆ phenylalkyl, C₇ toC₁₆ substituted phenylalkyl, phenyl, substituted phenyl, C₃ to C₇cycloalkyl, C₃ to C₇ substituted cycloalkyl, benzyl, and substitutedbenzyl; R³ is selected from the group consisting of a hydrogen atom, C₁to C₁₀ alkyl, C₁ to C₁₀ substituted alkyl, C₇ to C₁₆ phenylalkyl, C₇ toC₁₆ substituted phenylalkyl, phenyl, substituted phenyl, C₃ to C₇cycloalkyl, C₃ to C₇ substituted cycloalkyl, benzyl, and substitutedbenzyl; and R⁴ is selected from the group consisting of a hydrogen atom,C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₁ to C₁₀ substituted alkyl, C₂ toC₁₀ alkynyl, C₃ to C₇ substituted cycloalkyl, C₃ to C₇ cycloalkenyl, C₃to C₇ substituted cycloalkenyl, C₇ to C₁₆ phenylalkyl, C₇ to C₁₆substituted phenylalkyl, C₇ to C₁₆ phenylalkenyl and C₇ to C₁₆substituted phenylalkenyl, or a pharmaceutically-acceptable saltthereof.
 8. The compound of claim 7, wherein either R₁ or R₂ or both R₁and R₂ is not a hydrogen atom.
 9. The compound of claim 7, wherein: R¹is selected from the group consisting of methyl, benzyl, 2-butyl,2-methylpropyl, 2-propyl, 2-bromobenzyloxycarbonylbenzyl, ethyl,2-methylpropyl, propyl, butyl, 2-napthylmethyl, cyclohexylmethyl,4-fluorobenzyl, 4-chlorobenzyl, cyclohexyl, 4-ethoxybenzyl,4-iodobenzyl, and 4-methoxybenzyl; R² is selected from the groupconsisting of methyl, benzyl, 2-butyl, 2-methylpropyl, 2-propyl,2-bromobenzyloxycarbonylbenzyl, ethyl, propyl, butyl, 2-naphthylmethyl,methylsulfonylethyl, cyclohexylmethyl, 4-fluorobenzyl, 4-chlorobenzyl,cyclohexyl, 4-ethoxybenzyl, 4-iodobenzyl, and 4-methoxybenzyl; R³ isselected from the group consisting of methyl, benzyl, hydrogen,2-methylpropyl, propyl, butyl, cyclohexylmethyl, 4-ethoxybenzyl, and4-methoxybenzyl; and R⁴ is selected from the group consisting of1-phenyl-1-cyclopropyl, 1-phenylpropyl, 2-phenylpropyl, m-xylyl,3-fluorobenzyl, 3-bromobenzyl, 3-trifluoromethylbenzyl, p-xylyl,3-methoxybenzyl, 4-bromobenzyl, 4-methoxybenzyl, 4-ethoxybenzyl,1-(4-isobutylphenyl)ethyl, 3,4-dichlorobenzyl, 3-(3,4-dimethoxy)ethyl,4-biphenylmethyl, 1-phenylpropen-2-yl, 2-trifluoromethylstryl,3,4-dimethoxybenzyl, 3,4-dihydroxybenzyl, 2-methoxystyryl, phenyl,4-chlorostyryl, 3-methoxyphenyl, 4-isopropylphenyl, 4-vinylphenyl,4-fluorophenyl, 4-bromophenyl, 3,4-dimethoxystyryl, trans-styryl,3,4-dimethylphenyl, 3-fluoro-4-methylphenyl, 3-bromo-4-methylphenyl,3-iodo-4-methylphenyl, 3,4-dichlorophenyl, 4-biphenyl,3,4-difluorophenyl, m-tolyl, benzyl, phenethyl,3-methoxy-4-methylphenyl, 3-phenylpropyl, 3,4-dimethoxyphenyl,4-ethyl-4′-biphenyl, 3,4,5-trimethoxyphenyl, propyl, hexyl, 2-propyl,(+/−)-2-butyl, isobutyl, 2-methylbutyl, isovaleryl, p-tolyl, p-anisyl,cyclohexyl, cyclohexylmethyl, cyclohexylpropyl, cycloheptyl, methyl,2-methylcyclopropyl, cyclobutyl, cyclopentyyl, cyclopentylethyl,2-furyl, cyclohexylethyl, 4-methylcyclohexyl, 4-t-butylcyclohexyl,4-methylcyclohexylmethyl, but-2-en-1-yl, 2-norbornylmethyl, and2-thienyl.
 10. The compound of claim 1, wherein: R¹ is selected from thegroup consisting of methyl, benzyl, 2-butyl,N-methyl,N-thiocarbonylimidazole-aminobutyl, 2-methylpropyl,methylsulfinylethyl, guanidinopropyl, 2-propyl, 4-hydroxybenzyl, ethyl,dimethyl, propyl, butyl, N-methyl,N-thiocarbonylimidazole-aminopropyl,2-naphthylmethyl, cyclohexylmethyl, methylsulfonylethyl, 4-nitrobenzyl,4-chlorobenzyl, 4-fluorobenzyl,N-ethyl,N-thiocarbonylimidazole-aminobutyl, 3-pyridylmethyl, cyclohexyl,tert-butyl, N-methyl,N-thiocarbonylimidazole-4-aminobenzyl,4-ethoxybenzyl, 4-iodobenzyl, 4-methoxybenzyl andN-(methyl)indol-3-ylmethyl; R² is selected from the group consisting ofmethyl, benzyl, 2-butyl, N-methyl,N-thiocarbonylimidazole-aminobutyl,2-methylpropyl, methylsulfinylethyl, guanidinopropyl, 2-propyl,4-hydroxybenzyl, ethyl, propyl, butyl,N-methyl,N-thiocarbonylimidazole-aminopropyl, 2-naphthylmethyl,cyclohexylmethyl, methylsulfonylethyl, 4-nitrobenzyl, 4-chlorobenzyl,4-fluorobenzyl, N-ethyl,N-thiocarbonylimidazole-aminobutyl,3-pyridylmethyl, cyclohexyl, tert-butyl,N-methyl,N-thiocarbonylimidazole-4-aminobenzyl, 4-ethoxybenzyl,hydroxyethyl, 4-iodobenzyl, 4-methoxybenzyl and indol-3-ylmethyl; and R³is selected from the group consisting of 3-phenylbutyl, m-toluylethyl,3-fluorophenylethyl, p-toluylethyl, 4-fluorophenylethyl,3-methoxyphenylethyl, 4-methoxyphenylethyl, 4-ethoxyphenylethyl,3-(3,4-dimethoxyphenyl)propyl, 4-biphenylethyl,3,4-dimethoxyphenylethyl, phenylethyl, 3-phenylpropyl, 4-phenylbutyl,butyl, heptyl, isobutyryl, (+/−)-2-methylbutyl, isovaleryl,3-methylvaleryl, 4-methylvaleryl, (tert-butyl)ethyl, cyclohexylmethyl,cyclohexylethyl, cyclohexylbutyl, cycloheptylmethyl, 2-hydroxypropyl,ethyl, cyclobutylmethyl, cyclopentylmethyl, 3-cyclopentylpropyl,cyclohexylpropyl, 4-methyl-1-cyclohexylmethyl,4-(tert-butyl)-1-cyclohexylmethyl, 2-norbornylethyl, 1-adamantylethyl,2-ethylbutyl, 3,3-diphenylpropyl, 2-methyl-4-nitro-1-imidazolylpropyl,cyclopentylethyl, 3-indolylethyl and 2,4-dinitrophenylethyl.
 11. Thecompound of claim 1, wherein: R¹ is selected from the group consistingof methyl, benzyl, 2-butyl, N-methyl,N-thiocarbonylimidazole-aminobutyl,2-methylpropyl, methylsulfinylethyl, guanidinopropyl, 2-propyl,4-hydroxybenzyl, ethyl, dimethyl, propyl, butyl,N-methyl,N-thiocarbonylimidazole-aminopropyl, 2-naphthylmethyl,cyclohexylmethyl, methylsulfonylethyl, 4-nitrobenzyl, 4-chlorobenzyl,4-fluorobenzyl, N-ethyl,N-thiocarbonylimidazole-aminobutyl,3-pyridylmethyl, cyclohexyl, tert-butyl,N-methyl,N-thiocarbonylimidazole-4-aminobenzyl, 4-ethoxybenzyl,4-iodobenzyl, 4-methoxybenzyl and N-(methyl)indol-3-ylmethyl; R² isselected from the group consisting of benzyl, 2-butyl,N-methyl,N-thiocarbonylimidazole-aminobutyl, 2-methylpropyl,methylsulfinylethyl, guanidinopropyl, 2-propyl, 4-hydroxybenzyl, ethyl,propyl, butyl, N-methyl,N-thiocarbonylimidazole-aminopropyl,2-naphthylmethyl, cyclohexylmethyl, methylsulfonylethyl, 4-nitrobenzyl,4-chlorobenzyl, 4-fluorobenzyl,N-ethyl,N-thiocarbonylimidazole-aminobutyl, 3-pyridylmethyl, cyclohexyl,tert-butyl, N-methyl,N-thiocarbonylimidazole-4-aminobenzyl,4-ethoxybenzyl, hydroxyethyl, 4-iodobenzyl, 4-methoxybenzyl andindol-3-ylmethyl; and R³ is selected from the group consisting of3-phenylbutyl, m-toluylethyl, 3-fluorophenylethyl, p-toluylethyl,4-fluorophenylethyl, 3-methoxyphenylethyl, 4-methoxyphenylethyl,4-ethoxyphenylethyl, 3-(3,4-dimethoxyphenyl)propyl, 4-biphenylethyl,3,4-dimethoxyphenylethyl, phenylethyl, 3-phenylpropyl, 4-phenylbutyl,butyl, heptyl, isobutyryl, (+/−)-2-methylbutyl, isovaleryl,3-methylvaleryl, 4-methylvaleryl, (tert-butyl)ethyl, cyclohexylmethyl,cyclohexylethyl, cyclohexylbutyl, cycloheptylmethyl, 2-hydroxypropyl,ethyl, cyclobutylmethyl, cyclopentylmethyl, 3-cyclopentylpropyl,cyclohexylpropyl, 4-methyl-1-cyclohexylmethyl,4-(tert-butyl)-1-cyclohexylmethyl, 2-norbornylethyl, 1-adamantylethyl,2-ethylbutyl, 3,3-diphenylpropyl, 2-methyl-4-nitro-1-imidazolylpropyl,cyclopentylethyl, 3-indolylethyl and 2,4-dinitrophenylethyl.